Therapeutic antibodies and their uses

ABSTRACT

The present invention relates to antibodies, e.g., full length antibodies or antigen binding fragments thereof, that specifically bind to BCMA (B-Cell Maturation Antigen) and/or CD3 (Cluster of Differentiation 3). The invention also relates to antibody conjugates (e.g., antibody-drug-conjugates) comprising the BCMA antibodies, compositions comprising the BCMA antibodies, and methods of using the BCMA antibodies and their conjugates for treating conditions associated with cells expressing BCMA (e.g., cancer or autoimmune disease). The invention further relates to heteromultimeric antibodies that specifically bind to CD3 and a tumor cell antigen, (e.g., bispecific antibodies that specifically bind to CD3 and BCMA). Compositions comprising such heteromultimeric antibodies, methods for producing and purifying such heterodimeric antibodies, and their use in diagnostics and therapeutics are also provided.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/878,344, filed Jan. 23, 2018, which is a divisional of U.S.application Ser. No. 15/085,644, which was filed on Mar. 30, 2016, nowU.S. Pat. No. 9,969,809, which claims the benefits of U.S. ProvisionalApplication No. 62/146,843 filed Apr. 13, 2015, U.S. ProvisionalApplication No. 62/146,504 filed Apr. 13, 2015, and U.S. ProvisionalApplication No. 62/301,582 filed Feb. 29, 2016, all of which are herebyincorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled “PC72209C_SEQListing_ST25.txt”created on Jun. 27, 2018 and having a size of 294 KB. The sequencelisting contained in this .txt file is part of the specification and isincorporated herein by reference in its entirety.

FIELD

The present invention relates to antibodies, e.g., full lengthantibodies or antigen binding fragments thereof, that specifically bindto BCMA (B-Cell Maturation Antigen) and/or CD3 (Cluster ofDifferentiation 3). The invention also relates to antibody conjugates(e.g., antibody-drug-conjugates) comprising the BCMA antibodies,compositions comprising the BCMA antibodies, and methods of using theBCMA antibodies and their conjugates for treating conditions associatedwith cells expressing BCMA (e.g., cancer or autoimmune disease). Theinvention further relates to heteromultimeric antibodies thatspecifically bind to CD3 and a tumor cell antigen, (e.g., bispecificantibodies that specifically bind to CD3 and BCMA). Compositionscomprising such heteromultimeric antibodies, methods for producing andpurifying such heterodimeric antibodies, and their use in diagnosticsand therapeutics are also provided.

BACKGROUND

B-cell maturation antigen (BCMA, CD269, or TNFRSF17) is a member of thetumor necrosis factor receptor (TNFR) superfamily. BCMA was identifiedin a malignant human T cell lymphoma containing a t(4;16) translocation.The gene is selectively expressed in the B-cell lineage with the highestexpression in plasma blasts and plasma cells, antibody secreting cells.BCMA binds two ligands, B-cell activation factor (BAFF) (also calledB-lymphocyte stimulator (BLyS) and APOL-related leukocyte expressedligand (TALL-1)) and a proliferation-inducing ligand (APRIL) withaffinity of 1 uM and 16 nM, respectively. Binding of APRIL or BAFF toBCMA promotes a signaling cascade involving NF-kappa B, Elk-1, c-JunN-terminal kinase and the p38 mitogen-activated protein kinase, whichproduce signals for cell survival and proliferation.

BCMA is also expressed on malignant B cells and several cancers thatinvolve B lymphocytes including multiple myeloma, plasmacytoma,Hodgkin's Lymphoma, and chronic lymphocytic leukemia. In autoimmunediseases where plasmablasts are involved such as systemic lupuserythematosus (SLE) and rheumatoid arthritis, BCMA expressingantibody-producing cells secrete autoantibodies that attack self.

In the case of multiple myeloma, about 24,000 new cases are newlydiagnosed in the United States each year, and this number representsabout 15% of the newly diagnosed hematological cancers in the UnitedStates. An average of 11,000 deaths result from multiple myeloma eachyear, and the average 5-year survival rate is about 44%, with mediansurvival of 50-55 months. Current treatment for multiple myeloma isfocused on plasma cells apoptosis and/or decreasing osteoclast activity(e.g., chemotherapy, thalidomide, lenalidomide, bisphosphonates, and/orproteasome inhibitors such as bortezomib (VELCADE®) or carfilzomib).However, multiple myeloma remains an incurable disease, and almost allpatients have developed resistance to these agents and eventuallyrelapse. Accordingly, an alternative treatment to multiple myeloma, suchas using an anti-BCMA antagonist including antibodies and otherimmunotherapeutic agents (e.g. bispecific antibodies or antibody-drugconjugates), would make a superior therapeutic agent.

SUMMARY

The invention disclosed herein is directed to therapeutic antibodiesthat bind to BCMA and/or CD3. Antibody conjugates (e.g., antibody-drugconjugates) comprising BCMA are also provided. Further, theheteromultimeric antibodies (e.g., bispecific antibodies) thatspecifically bind to CD3 and a tumor cell antigen (e.g., bispecificantibodies that specifically bind to CD3 and BCMA) are also provided.

In one aspect, the invention provides an isolated antibody, or anantigen binding fragment thereof, which specifically binds to B-CellMaturation Antigen (BCMA), wherein the antibody comprises (a) a heavychain variable (VH) region comprising (i) a VH complementary determiningregion one (CDR1) comprising the sequence SYX₁MX₂, wherein X₁ is A or P;and X₂ is T, N, or S (SEQ ID NO: 301), GFTFX₁SY, wherein X₁ is G or S(SEQ ID NO: 302), or GFTFX₁SYX₂MX₃, wherein X₁ is G or S, X₂ is A or P;and X₃ is T, N, or S (SEQ ID NO: 303); (ii) a VH CDR2 comprising thesequence AX₁X₂X₃X₄GX₅X₆X₇X₈YADX₉X₁₀KG, wherein X₁ is I, V, T, H, L, A,or C; X₂ is S, D, G, T, I, L, F, M, or V; X₃ is G, Y, L, H, D, A, S, orM; X₄ is S, Q, T, A, F, or W; X₅ is G or T; X₆ is N, S, P, Y, W, or F;X₇ is S, T, I, L, T, A, R, V, K, G, or C; X₈ is F, Y, P, W, H, or G; X₉is V, R, or L; and X₁₀ is G or T (SEQ ID NO: 305), or X₁X₂X₃X₄X₅X₆,wherein X₁ is S, V, I, D, G, T, L, F, or M; X₂ is G, Y, L, H, D, A, S,or M; X₃ is S, G, F, or W; X₄ is G or S; X₅ is G or T; and X₆ is N, S,P, Y, or W (SEQ ID NO: 306); and iii) a VH CDR3 comprising the sequenceVSPIX₁X₂X₃X₄, wherein X₁ is A or Y; X₂ is A or S; and X₃ is G, Q, L, P,or E (SEQ ID NO: 307), or YWPMX₁X₂, wherein X₁ is D, S, T, or A; and X₂is I, S, L, P, or D (SEQ ID NO: 308); and/or (b) a light chain variable(VL) region comprising (i) a VL CDR1 comprising the sequenceX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂, wherein X₁ is R, G, W, A, or C; X₂ is A, P,G, L, C, or S; X₃ is S, G, or R; X₄ is Q, C, E, V, or I; X₅ is S, P, G,A, R, or D; X₆ is V, G, I, or L; X₇ is S, E, D, P, or G; X₈ is S, P, F,A, M, E, V, N, D, or Y; X₉ is I, T, V, E, S, A, M, Q, Y, H, R, or F; X₁₀is Y or F; X₁₁ is L, W, or P; and X₁₂ is A, S, or G (SEQ ID NO: 309);(ii) a VL CDR2 comprising the sequence X₁ASX₂RAX₃, wherein X₁ is G or D;X₂ is S or I; and X₃ is T or P (SEQ ID NO: 310); and (iii) a VL CDR3comprising the sequence QQYX₁X₂X₃PX₄T, wherein X₁ is G, Q, E, L, F, A,S, M, K, R, or Y; X₂ is S, R, T, G, V, F, Y, D, A, H, V, E, K, or C; X₃is W, F, or S; and X₄ is L or I (SEQ ID NO: 311), or QQYX₁X₂X₃PX₄,wherein X₁ is G, Q, E, L, F, A, S, M, R, K, or Y; X₂ is S, R, T, G, R,V, D, A, H, E, K, C, F, or Y; X₃ is W, S, or F; and X₄ is L or I (SEQ IDNO: 312).

In another aspect, the invention provides an isolated antibody, or anantigen binding fragment thereof, which specifically binds to BCMA,wherein the antibody comprises: a VH region comprising a VH CDR1, VHCDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 2, 3, 7, 8, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 37, 39, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 83, 87, 92, 95, 97,99, 101, 104, 106, 110, 112, 114, 118, 120, 122, 125, 127, 313, 314,363, or 365; and/or a VL region comprising VL CDR1, VL CDR2, and VL CDR3of the VL sequence shown in SEQ ID NO: 1, 4, 5, 6, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 34, 36, 38, 40, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 317, 81,82, 84, 85, 86, 88, 89, 90, 91, 93, 94, 96, 98, 100, 102, 103, 105, 107,108, 109, 111, 113, 115, 116, 117, 119, 121, 123, 124, 126, 128, 315,316, or 364. In some embodiments, the VH region comprises (i) a VH CDR1comprising SEQ ID NO:150, 151, 152, 156, or 157; (ii) a VH CDR2comprising SEQ ID NO: 169, 154, 194, 159, 195, 196, 162, 158, 198, 177,178, 199, 200, 201, 202, 203, 204, 206, 207, 208, 172, 203, or 204; and(iii) a VH CDR3 comprising SEQ ID NO: 155, 161, 197, 205, or 164; and/orwherein the VL region comprises (i) a VL CDR1 comprising SEQ ID NO: 209,271, 273, 275, 251, 277, 260, 279, 245, 283, 285, 287, 290, 292, 235,297, or 299; (ii) a VL CDR2 comprising SEQ ID NO: 221; and (iii) a VLCDR3 comprising SEQ ID NO: 225, 272, 274, 276, 278, 280, 281, 282, 284,286, 288, 289, 291, 293, 294, 229, 296, 298, or 300. In someembodiments, the VH region comprises the sequence shown in SEQ ID NO:112 or a variant with one or several conservative amino acidsubstitutions in residues that are not within a CDR and/or the VL regioncomprises the amino acid sequence shown in SEQ ID NO: 38 or a variantthereof with one or several amino acid substitutions in amino acids thatare not within a CDR. In some embodiments, the antibody comprises alight chain comprising the sequence shown in SEQ ID NO: 357 and a heavychain comprising the sequence shown in SEQ ID NO: 358. In someembodiments, the antibody comprises a VH region produced by theexpression vector with ATCC Accession No. PTA-122094. In someembodiments, the antibody comprises a VL region produced by theexpression vector with ATCC Accession No. PTA-122093.

In another aspect, the invention provides an isolated antibodycomprising an acyl donor glutamine-containing tag engineered at aspecific site of the BCMA antibody of the present invention. In someembodiments, the tag comprises an amino acid sequence selected from thegroup consisting of Q, LQG, LLQGG (SEQ ID NO:318), LLQG (SEQ ID NO:454),LSLSQG (SEQ ID NO: 455), GGGLLQGG (SEQ ID NO: 456), GLLQG (SEQ ID NO:457), LLQ, GSPLAQSHGG (SEQ ID NO: 458), GLLQGGG (SEQ ID NO: 459), GLLQGG(SEQ ID NO: 460), GLLQ (SEQ ID NO: 461), LLQLLQGA (SEQ ID NO: 462),LLQGA (SEQ ID NO: 463), LLQYQGA (SEQ ID NO: 464), LLQGSG (SEQ ID NO:465), LLQYQG (SEQ ID NO: 466), LLQLLQG (SEQ ID NO: 467), SLLQG (SEQ IDNO: 468), LLQLQ (SEQ ID NO: 469), LLQLLQ (SEQ ID NO: 470), LLQGR (SEQ IDNO: 471), LLQGPP (SEQ ID NO: 472), LLQGPA (SEQ ID NO: 473), GGLLQGPP(SEQ ID NO: 474), GGLLQGA (SEQ ID NO: 475), LLQGPGK (SEQ ID NO: 476),LLQGPG (SEQ ID NO: 477), LLQGP (SEQ ID NO: 478), LLQP (SEQ ID NO: 479),LLQPGK (SEQ ID NO: 480), LLQAPGK (SEQ ID NO: 481), LLQGAPG (SEQ ID NO:482), LLQGAP (SEQ ID NO: 483), and LLQLQG (SEQ ID NO: 484).

In one variation, the invention provides an isolated antibody comprisingan acyl donor glutamine-containing tag and an amino acid modification atposition 222, 340, or 370 of the BCMA antibody of the present invention.In some embodiments, the amino acid modification is a substitution fromlysine to arginine.

In some embodiments, the BCMA antibody of the present invention furthercomprises a linker. In some embodiments, the linker is selected from thegroup consisting of Ac-Lys-Gly (acetyl-lysine-glycine), aminocaproicacid, Ac-Lys-β-Ala (acetyl-lysine-β-alanine), amino-PEG2 (polyethyleneglycol)-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys-Val-Cit-PABC(acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl),amino-PEG6-C2-Val-Cit-PABC, aminocaproyl-Val-Cit-PABC,[(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC,[(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC,putrescine, and Ac-Lys-putrescine.

In another aspect, the invention provides a conjugate of the BCMAantibody or the antigen binding fragment as described herein, whereinthe antibody or the antigen binding fragment is conjugated to an agent,wherein the agent is selected from the group consisting of a cytotoxicagent, an immunomodulating agent, an imaging agent, a therapeuticprotein, a biopolymer, and an oligonucleotide. In some embodiments, theagent is a cytotoxic agent including, but not limited to, ananthracycline, an auristatin, a camptothecin, a combretastatin, adolastatin, a duocarmycin, an enediyne, a geldanamycin, anindolino-benzodiazepine dimer, a maytansine, a puromycin, apyrrolobenzodiazepine dimer, a taxane, a vinca alkaloid, a tubulysin, ahemiasterlin, a spliceostatin, a pladienolide, and stereoisomers,isosteres, analogs, or derivatives thereof. For example, the cytotoxicagent is MMAD (Monomethyl Auristatin D), 0101(2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide),3377(N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxyl-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide),0131(2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide),or0121(2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide).

In some embodiments, the present invention provides a conjugatecomprising the formula: antibody-(acyl donor glutamine-containingtag)-(linker)-(cytotoxic agent). In some embodiments, the acyl donorglutamine-containing tag comprises an amino acid sequence LLQG (SEQ IDNO: 319) and/or GGLLQGPP (SEQ ID NO: 339) and wherein the linkercomprises acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl oramino-PEG6-C2. In some embodiments, the conjugate is selected from thegroup consisting of 1) antibody-GGLLQGPP (SEQ ID NO:339)-(acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl(AcLys-VC-PABC))-0101; 2) antibody-LLQG (SEQ ID NO:319)-amino-PEG6-C2-0131; and 3) antibody-LLQG (SEQ ID NO:319)-amino-PEG6-C2-3377. In some embodiments, the conjugate furthercomprises an amino acid substitution from lysine to arginine at antibodyposition 222. In some embodiments, the conjugate further comprises aminoacid substitutions at antibody position N297Q or N297A.

In another aspect, provided is a method of producing the BCMA antibodyas described herein, comprising culturing the host cell under conditionsthat result in production of the BCMA antibody, and isolating the BCMAantibody from the host cell or culture.

In another aspect, the invention provides a use of the BCMA antibodiesor the BCMA antibody conjugates as described herein in the manufactureof a medicament for treating a condition (e.g., cancer or autoimmunedisorder) associated with BCMA expression. In some embodiments, providedis a use of the BCMA antibodies or the BCMA antibody conjugates asdescribed herein in the manufacture of a medicament for inhibiting tumorgrowth or progression. In some embodiments, provided is a use of theBCMA antibodies or the BCMA antibody conjugates as described herein inthe manufacture of a medicament for inhibiting metastasis of malignantcells expressing BCMA. In some embodiments, provided is a use of theBCMA antibodies or the BCMA antibody conjugates as described herein inthe manufacture of a medicament for inducing tumor regression.

In another aspect, the invention provides an isolated antibody, or anantigen binding fragment thereof, which specifically binds to CD3,wherein the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of the VHsequence shown in SEQ ID NO: 320, 322, 324, 326, 328, 330, 345, 347,349, 351, 444, 354, 356, 378, 442, 380, 382, 384 386, 388, 390, 392,394, 396, 398, or 400; and/or a light chain variable (VL) regioncomprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQID NO: 319, 321, 323, 325, 327, 329, 344, 346, 348, 350, 352, 355, 377,443, 445, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, or 399. Insome embodiments, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3of the VH sequence shown in SEQ ID NO: 324 or 388; and/or a light chainvariable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VLsequence shown in SEQ ID NO: 323 or 387. In some embodiments, the VHregion comprises (i) a VH complementarity determining region one (CDR1)comprising the sequence shown in SEQ ID NO: 331, 332, 333, 401, 402,403, 407, 408, 415, 416, 418, 419, 420, 424, 425, 426, 446, 447, or 448(ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 334, 336,337, 338, 339, 404, 405, 409, 410, 411, 412, 413, 414, 417, 418, 421,422, 427, 428, 449, or 450; and iii) a VH CDR3 comprising the sequenceshown in SEQ ID NO: 335, 406, 423, 429, or 451; and/or a light chainvariable (VL) region comprising (i) a VL CDR1 comprising the sequenceshown in SEQ ID NO: 340, 343, 430, 431, 435, or 440, 441; (ii) a VL CDR2comprising the sequence shown in SEQ ID NO: 341, 433, 452, or 436; and(iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 342, 432,434, 437, 438, 439, 446, or 453. In some embodiments, the VH regioncomprises (i) a VH complementarity determining region one (CDR1)comprising the sequence shown in SEQ ID NO: 331, 332, 333, 401, 407, or408 (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 336, 404,405, or 417; and iii) a VH CDR3 comprising the sequence shown in SEQ IDNO: 335 or 406; and/or a light chain variable (VL) region comprising (i)a VL CDR1 comprising the sequence shown in SEQ ID NO: 343 or 441; (ii) aVL CDR2 comprising the sequence shown in SEQ ID NO: 341 or 436; and(iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 342 or 439.In some embodiments, the antibody comprises a VH region produced by theexpression vector with ATCC Accession No. PTA-122513. In someembodiments, the antibody comprises a VL region produced by theexpression vector with ATCC Accession No. PTA-122512.

In another aspect, provided is an isolated antibody which specificallybinds to CD3 and competes with the anti-CD3 antibody of the presentinvention as described herein.

In another aspect, the invention provides a bispecific antibody whereinthe bispecific antibody is a full-length human antibody, comprising afirst antibody variable domain of the bispecific antibody capable ofrecruiting the activity of a human immune effector cell by specificallybinding to an effector antigen located on the human immune effectorcell, and comprising a second antibody variable domain of the bispecificantibody capable of specifically binding to a target antigen, whereinthe first antibody variable domain comprises a heavy chain variable (VH)region comprising a VH CDR1, VH CDR2, and VH CDR3 of the VH sequenceshown in SEQ ID NO: 320, 322, 324, 326, 328, 330, 345, 347, 349, 351,444, 354, 356, 378, 442, 380, 382, 384 386, 388, 390, 392, 394, 396,398, or 400; and/or a light chain variable (VL) region comprising VLCDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 319,321, 323, 325, 327, 329, 344, 346, 348, 350, 352, 355, 377, 443, 445,379, 381, 383, 385, 387, 389, 391, 393, 395, 397, or 399. In someembodiments, the first antibody variable domain comprises a heavy chainvariable (VH) region comprising (i) a VH CDR1 comprising the sequenceshown in SEQ ID NO: 331, 332, 333, 401, 402, 403, 407, 408, 415, 416,418, 419, 420, 424, 425, 426, 446, 447, or 448 (ii) a VH CDR2 comprisingthe sequence shown in SEQ ID NO: 334, 336, 337, 338, 339, 404, 405, 409,410, 411, 412, 413, 414, 417, 418, 421, 422, 427, 428, 449, or 450; andiii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 335, 406,423, 429, or 451; and/or a light chain variable (VL) region comprising(i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 340, 343, 430,431, 435, or 440, 441; (ii) a VL CDR2 comprising the sequence shown inSEQ ID NO: 341, 433, 452, or 436; and (iii) a VL CDR3 comprising thesequence shown in SEQ ID NO: 342, 432, 434, 437, 438, 439, 446, or 453.In some embodiments, the first antibody variable domain comprises aheavy chain variable (VH) region comprising a VH CDR1, VH CDR2, and VHCDR3 of the VH sequence shown in SEQ ID NO: 324 or 388; and/or a lightchain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 ofthe VL sequence shown in SEQ ID NO: 323 or 387; and the second antibodyvariable domain comprises a heavy chain variable (VH) region comprisinga VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO:112; and/or a light chain variable (VL) region comprising VL CDR1, VLCDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 38.

In some embodiments, the second antibody variable domain comprises (a) aheavy chain variable (VH) region comprising (i) a VH complementaritydetermining region one (CDR1) comprising the sequence SYX₁MX₂, whereinX₁ is A or P; and X₂ is T, N, or S (SEQ ID NO: 301), GFTFX₁SY, whereinX₁ is G or S (SEQ ID NO: 302), or GFTFX₁SYX₂MX₃, wherein X₁ is G or S,X₂ is A or P; and X₃ is T, N, or S (SEQ ID NO: 303); (ii) a VH CDR2comprising the sequence AX₁X₂X₃X₄GX₅X₆X₇X₈YADX₉X₁₀KG, wherein X₁ is I,V, T, H, L, A, or C; X₂ is S, D, G, T, I, L, F, M, or V; X₃ is G, Y, L,H, D, A, S, or M; X₄ is S, Q, T, A, F, or W; X₅ is G or T; X₆ is N, S,P, Y, W, or F; X₇ is S, T, I, L, T, A, R, V, K, G, or C; X₅ is F, Y, P,W, H, or G; X₉ is V, R, or L; and X₁₀ is G or T (SEQ ID NO: 305), orX₁X₂X₃X₄X₅X₆, wherein X₁ is S, V, I, D, G, T, L, F, or M; X₂ is G, Y, L,H, D, A, S, or M; X₃ is S, G, F, or W; X₄ is G or S; X₅ is G or T; andX₆ is N, S, P, Y, or W (SEQ ID NO: 306); and iii) a VH CDR3 comprisingthe sequence VSPIX₁X₂X₃X₄ wherein X₁ is A or Y; X₂ is A or S; and X₃ isG, Q, L, P, or E (SEQ ID NO: 307), or YWPMX₁X₂, wherein X₁ is D, S, T,or A; and X₂ is I, S, L, P, or D (SEQ ID NO: 308); and/or (b) a lightchain variable (VL) region comprising (i) a VL CDR1 comprising thesequence X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂, wherein X₁ is R, G, W, A, or C; X₂is A, P, G, L, C, or S; X₃ is S, G, or R; X₄ is Q, C, E, V, or I; X₅ isS, L, P, G, A, R, or D; X₆ is V, G, or I; X₇ is S, E, D, or P; X₅ is S,P, F, A, M, E, V, N, D, or Y; X₉ is I, T, V, E, S, A, M, Q, Y, H, or R;X₁₀ is Y or F; X₁₁ is L, W, or P; and X₁₂ is A, S, or G (SEQ ID NO:309); (ii) a VL CDR2 comprising the sequence X₁ASX₂RAX₃, wherein X₁ is Gor D; X₂ is S or I; and X₃ is T or P (SEQ ID NO: 310); and (iii) a VLCDR3 comprising the sequence QQYX₁X₂X₃PX₄T, wherein X₁ is G, Q, E, L, F,A, S, M, K, R, or Y; X₂ is S, R, T, G, V, F, Y, D, A, H, V, E, K, or C;X₃ is W, F, or S; and X₄ is L or I (SEQ ID NO: 311), or QQYX₁X₂X₃PX₄,wherein X₁ is G, Q, E, L, F, A, S, M, R, K, or Y; X₂ is S, R, T, G, R,V, D, A, H, E, K, C, F, or Y; X₃ is W, S, or F; and X₄ is L or I (SEQ IDNO: 312). In some embodiments, the second antibody variable domaincomprises a heavy chain variable (VH) region comprising (i) a VH CDR1comprising the sequence shown in SEQ ID NO:150, 151, 152, 156, 157, 348,349, 353, 354, or 355; (ii) a VH CDR2 comprising the sequence shown inSEQ ID NO: 169, 154, 194, 159, 195, 196, 162, 158, 198, 177, 178, 199,200, 201, 202, 203, 204, 206, 207, 208, 172, 203, 204, 350, 351, 356 or357; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO:155, 161, 197, 205, 164, or 352, or 358; and/or wherein the light chainvariable (VL) region comprises (i) a VL CDR1 comprising the sequenceshown in SEQ ID NO: 209, 271, 273, 275, 251, 277, 260, 279, 245, 283,285, 287, 290, 292, 235, 297, 299, or 361; (ii) a VL CDR2 comprising thesequence shown in SEQ ID NO: 221, 359 or 362; and (iii) a VL CDR3comprising the sequence shown in SEQ ID NO: 211, 225, 272, 274, 276,278, 280, 281, 282, 284, 286, 288, 289, 291, 293, 294, 229, 296, 298,300 or 360.

In some embodiments, (a) the first antibody variable domain comprises aheavy chain variable (VH) region comprising (i) a VH complementaritydetermining region one (CDR1) comprising the sequence shown in SEQ IDNO: 331, 332, 333, 401, 407, or 408 (ii) a VH CDR2 comprising thesequence shown in SEQ ID NO: 336, 417, 404, or 405; and iii) a VH CDR3comprising the sequence shown in SEQ ID NO: 335 or 406; and/or a lightchain variable (VL) region comprising (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 343 or 441; (ii) a VL CDR2 comprising thesequence shown in SEQ ID NO: 341 or 436; and (iii) a VL CDR3 comprisingthe sequence shown in SEQ ID NO: 342 or 439; and (b) the second antibodyvariable domain comprises a heavy chain VH region comprising a heavychain variable (VH) region comprising (i) a VH CDR1 comprising thesequence shown in SEQ ID NO: 151, 156, or 157; (ii) a VH CDR2 comprisingthe sequence shown in SEQ ID NO: 158 or 159; and (iii) a VH CDR3comprising SEQ ID NO: 155; and/or wherein the light chain variable (VL)region comprises (i) a VL CDR1 comprising the sequence shown in SEQ IDNO: 209; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 221;and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 225.

In some embodiments, both the first and the second antibody variabledomains of the bispecific antibody comprise amino acid modifications atpositions 223, 225, and 228 in the hinge region and at position 409 or368 (EU numbering scheme) in the CH3 region of a human IgG2 (SEQ ID NO:493). In some embodiments, the bispecific antibody as described hereinfurther comprises an amino acid modification at position 265 of thehuman IgG2.

In another aspect, the invention provides pharmaceutical compositionscomprising any of the antibodies (e.g., BCMA, CD3, or bispecific) or theconjugates thereof (e.g., BCMA antibody-drug conjugate) describedherein.

In another aspect, the invention also provides cell lines thatrecombinantly produce any of the antibodies (e.g., BCMA, CD3, orbispecific) or the conjugates thereof (e.g., BCMA antibody-drugconjugate) described herein.

In another aspect, the invention also provides nucleic acids encodingany of the antibodies (e.g., BCMA, CD3, or bispecific) or the conjugatesthereof (e.g., BCMA antibody-drug conjugate) described herein. Theinvention also provides nucleic acids encoding a heavy chain variableregion and/or a light chain variable region of any of these antibodiesdescribed herein.

The invention also provides kits comprising an effective amount of anyof the antibodies (e.g., BCMA, CD3, or bispecific) or the conjugatesthereof (e.g., BCMA antibody-drug conjugate) described herein.

The invention also provides methods of treating a condition (e.g., tumorgrowth/progression inhibition; metastasis of malignant cells expressingBCMA inhibition; tumor regression induction in subjects with malignantcells expressing BCMA) in subjects in need thereof comprising providingthe isolated antibodies (e.g., BCMA) or binding fragments, bispecificantibodies (BCMA-CD3 bispecifics), or the conjugates thereof (e.g., BCMAantibody-drug conjugates) thereof described herein and administeringsaid antibodies or conjugates to said subject.

Also provided are methods of treating a condition associated withmalignant cells expressing a tumor antigen in a subject comprisingadministering to a subject in need thereof an effective amount of thepharmaceutical compositions of the invention. In some embodiments, thecondition is cancer. In some embodiments, the cancer is a B-cell relatedcancer selecting from the group consisting of multiple myeloma,malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocytepredominant Hodgkin's lymphoma, Kahler's disease and Myelomatosis,plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia,hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloidleukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocyticleukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma,Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, largecell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia,Waldenstrom's macroglobulienemia, diffuse large B cell lymphoma,follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphatictissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma,Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma,lymphoplasmactyic lymphoma, Waldenström macroglobulinemia, nodalmarginal zone B cell lymphoma, splenic marginal zone lymphoma,intravascular large B-cell lymphoma, primary effusion lymphoma,lymphomatoid granulomatosis, T cell/histiocyte-rich large B-celllymphoma, primary central nervous system lymphoma, primary cutaneousdiffuse large B-cell lymphoma (leg type), EBV positive diffuse largeB-cell lymphoma of the elderly, diffuse large B-cell lymphoma associatedwith inflammation, intravascular large B-cell lymphoma, ALK-positivelarge B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphomaarising in HHV8-associated multicentric Castleman disease, B-celllymphoma unclassified with features intermediate between diffuse largeB-cell lymphoma and Burkitt lymphoma, B-cell lymphoma unclassified withfeatures intermediate between diffuse large B-cell lymphoma andclassical Hodgkin lymphoma, and other B-cell related lymphoma. In someembodiments, the condition is an autoimmune disorder, such as systemiclupus erythematosus or rheumatoid arthritis.

In some embodiments, the antibodies described herein comprise a constantregion. In some embodiments, the antibodies described herein are of thehuman IgG1, IgG2 or IgG2Δa, IgG3, or IgG4 subclass. In some embodiments,the antibodies described herein comprise a glycosylated constant region.In some embodiments, the antibodies described herein comprise a constantregion having increased binding affinity to one or more human Fc gammareceptor(s).

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

FIG. 1A-FIG. 1D depict the double-referenced sensorgrams with fit curvesfor interactions between selected anti-BCMA antibodies of the presentinvention and human BCMA.

FIG. 2 depicts in vivo efficacy studies of various anti-BCMA ADCs in theMM1S orthotopic multiple myeloma model, includingP6E01_VHVL-AcLys-Val-Cit-PABC-Aur0101;P5A2_VHVL-AcLys-Val-Cit-PABC-Aur0101;P5C1_VHVL-AcLys-Val-Cit-PABC-Aur0101; P4G4-AcLys-Val-Cit-PABC-Aur0101;and P1A11-AcLys-Val-Cit-PABC-Aur0101. NNC is a negative control non-BCMAantibody. “LCQ05” and “LCQ04” correspond to glutamine-containingtransglutaminase tag SEQ ID NOs: 474 and 475, respectively.

FIG. 3 depicts in vivo efficacy of the anti-BCMA ADCs in the MM1Sorthotopic multiple myeloma model, including L3.PY/P6E01 antibodyconjugated with 1) H7c/N297A/K222R-amino-PEG6-C2-3377, 2)N297Q/K222R-AcLys-Val-Cit-PABC-0101, 3)LCQ05/K222R-AcLys-Val-Cit-PABC-0101, 4)H7c/N297A/K222R-amino-PEG6-C2-0131, and 5)N297Q/K222R/LCQ05-AcLys-Val-Cit-PABC-Aur0101. NNC is a control non-BCMAantibody. “LCQ05” and H7c correspond to glutamine-containingtransglutaminase tag SEQ ID NO: 474 and SEQ ID NO: 454, respectively

FIG. 4 also depicts in vivo efficacy of the anti-BCMA ADCs in the MM1Sorthotopic multiple myeloma model, including L3.PY/P6E01 antibodyconjugated with 1) H7c/N297A/K222R-amino-PEG6-C2-3377, 2)N297Q/K222R-AcLys-Val-Cit-PABC-Aur0101, 3)LCQ05/K222R-AcLys-Val-Cit-PABC-Aur0101, 4)H7c/N297A/K222R-amino-PEG6-C2-0131, and 5)N297Q/K222R/LCQ05-AcLys-Val-Cit-PABC-Aur0101. NNC is a control non-BCMAantibody (antibody-N297Q/K222R-AcLys-VC-PABC-0101). “LCQ05” and H7ccorrespond to glutamine-containing transglutaminase tag SEQ ID NO: 474and SEQ ID NO: 454, respectively.

FIG. 5 also depicts in vivo efficacy of an anti-BCMA ADC in the MM1Sorthotopic multiple myeloma model. Anti-BCMA antibody COMBO_Rd4_0.6nM-C29 (“Combo C29 DI) is conjugated toH7c/N297A/K222R-amino-PEG6-C2-131 at doses ranging from 0.1 mg/kg, 0.38mg/kg, 0.75 mg/kg, 1.5 mg/kg in comparison to NNC, a control non-BCMAantibody (antibody-N297Q/K222R-AcLys-VC-PABC-0101) at 3 mg/kg. H7ccorrespond to glutamine-containing transglutaminase tag SEQ ID NO: 454.

FIG. 6A-FIG. 6F depict the in vivo efficacy of an anti-CD3/anti-CD20bispecific antibody in cynomolgus monkeys. B cell depletion following asingle dose of bispecific antibody is shown as a percentage of prestudycounts.

FIG. 7A-FIG. 7F depict the in vivo efficacy of an anti-CD3/anti-CD20bispecific antibody in cynomolgus monkeys. CD8+ T cell kinetics weretracked following a single dose of bispecific antibody.

FIG. 8A and FIG. 8B depict the in vivo efficacy of an anti-CD3/anti-CD20bispecific antibody in cynomolgus monkeys. The effect of the monovalentCD3 antibody on T cell kinetics and proliferation was analyzed.

FIGS. 9A-FIG. 9D depict the in vivo efficacy of an anti-CD3/anti-CD20bispecific antibody in cynomolgus monkeys. The effect of anti-CD3 armaffinity on B cell depletion was analyzed.

FIGS. 10A and 10B show that the selected anti-CD3 antibodies hadThymidine incorporation reading on human and cynomolgus PBMC.

FIG. 11A-FIG. 11D show that all human anti-EpCam_h2B4 bispecificantibodies have cell killing activity on in vitro setting.

FIG. 12 shows that a single dose of human anti-BCMA/CD3 bispecificantibody resulted in tumor regression in a dose-dependent manner in anorthotopic MM1.S myeloma model.

FIG. 13 shows that two doses of human anti-BCMA/CD3 bispecific antibodyresulted in increased tumor regression in an orthotopic Molp8 myelomamodel.

FIG. 14 shows that anti-BCMA/CD3 bispecific antibody alone or incombination with standard of care for multiple myeloma (lenalidomide orbortezomib) is more efficacious than lenalidomide and bortezomibcombined in orthotopic Molp8 tumor model.

FIG. 15A-FIG. 15C, respectively, show that carfilzomib, lenalidomide,and doxorubicin do not have a negative effect on the function of theanti-BCMA/CD3 bispecific antibody on OPM2 cells as compared to theanti-BCMA/CD3 bispecific antibody alone.

FIG. 16 shows synergistic effects on the function of anti-BCMA/CD3bispecific antibody when combined with carfilzomib and lenalidomide incomparison to each molecule alone.

DETAILED DESCRIPTION

The invention disclosed herein provides antibodies and antibodyconjugates (e.g., antibody-drug conjugates) that specifically bind toBCMA (e.g., human BCMA). The invention also provides polynucleotidesencoding these antibodies and conjugates, compositions comprising theseantibodies and conjugates, and methods of making these antibodies andconjugates. Further, the invention disclosed herein provides antibodiesthat specifically bind to CD3 (e.g., human CD3) as well as heterodimericantibodies (e.g., bispecific antibodies) that specifically bind to CD3and a tumor antigen (e.g., BCMA). The invention also providespolynucleotides encoding these antibodies, compositions comprising theseantibodies, and methods of making and using these antibodies. Theinvention further provides methods for treating a condition associatedwith malignant BCMA expression in a subject, such as cancer orautoimmune disease, using the antibodies (e.g., BCMA, CD3, or bispecificantibody) or conjugates thereof (BCMA antibody-drug conjugates) asdescribed herein.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (AcademicPress, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty, ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Definitions

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain(ScFv) and domain antibodies (including, for example, shark and camelidantibodies), and fusion proteins comprising an antibody, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen recognition site. An antibody includes an antibody of any class,such as IgG, IgA, or IgM (or subclass thereof), and the antibody neednot be of any particular class. Depending on the antibody amino acidsequence of the constant region of its heavy chains, immunoglobulins canbe assigned to different classes. There are five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2. The heavy-chain constant regions that correspond tothe different classes of immunoglobulins are called alpha, delta,epsilon, gamma, and mu, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

The term “antigen binding fragment” or “antigen binding portion” of anantibody, as used herein, refers to one or more fragments of an intactantibody that retain the ability to specifically bind to a given antigen(e.g., BCMA or CD3). Antigen binding functions of an antibody can beperformed by fragments of an intact antibody. Examples of bindingfragments encompassed within the term “antigen binding fragment” of anantibody include Fab; Fab′; F(ab′)₂; an Fd fragment consisting of the VHand CH1 domains; an Fv fragment consisting of the VL and VH domains of asingle arm of an antibody; a single domain antibody (dAb) fragment (Wardet al., Nature 341:544-546, 1989), and an isolated complementaritydetermining region (CDR).

An antibody, an antibody conjugate, or a polypeptide that“preferentially binds” or “specifically binds” (used interchangeablyherein) to a target (e.g., BCMA protein or CD3 protein) is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecule is saidto exhibit “specific binding” or “preferential binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular cell or substance than it doeswith alternative cells or substances. An antibody “specifically binds”or “preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to a BCMA epitope or CD3 epitope is an antibodythat binds this epitope with greater affinity, avidity, more readily,and/or with greater duration than it binds to other BCMA epitopes,non-BCMA epitopes, CD3 epitopes, or non-CD3 epitopes. It is alsounderstood that by reading this definition, for example, an antibody (ormoiety or epitope) that specifically or preferentially binds to a firsttarget may or may not specifically or preferentially bind to a secondtarget. As such, “specific binding” or “preferential binding” does notnecessarily require (although it can include) exclusive binding.Generally, but not necessarily, reference to binding means preferentialbinding.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. As known in the art, the variableregions of the heavy and light chain each consist of four frameworkregions (FR) connected by three complementarity determining regions(CDRs) also known as hypervariable regions. The CDRs in each chain areheld together in close proximity by the FRs and, with the CDRs from theother chain, contribute to the formation of the antigen binding site ofantibodies. There are at least two techniques for determining CDRs: (1)an approach based on cross-species sequence variability (i.e., Kabat etal. Sequences of Proteins of Immunological Interest, (5th ed., 1991,National Institutes of Health, Bethesda Md.)); and (2) an approach basedon crystallographic studies of antigen-antibody complexes (Al-lazikaniet al., 1997, J. Molec. Biol. 273:927-948). As used herein, a CDR mayrefer to CDRs defined by either approach or by a combination of bothapproaches.

A “CDR” of a variable domain are amino acid residues within the variableregion that are identified in accordance with the definitions of theKabat, Chothia, the accumulation of both Kabat and Chothia, AbM,contact, and/or conformational definitions or any method of CDRdetermination well known in the art. Antibody CDRs may be identified asthe hypervariable regions originally defined by Kabat et al. See, e.g.,Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5thed., Public Health Service, NIH, Washington D.C. The positions of theCDRs may also be identified as the structural loop structures originallydescribed by Chothia and others. See, e.g., Chothia et al., Nature342:877-883, 1989. Other approaches to CDR identification include the“AbM definition,” which is a compromise between Kabat and Chothia and isderived using Oxford Molecular's AbM antibody modeling software (nowAccelrys®), or the “contact definition” of CDRs based on observedantigen contacts, set forth in MacCallum et al., J. Mol. Biol.,262:732-745, 1996. In another approach, referred to herein as the“conformational definition” of CDRs, the positions of the CDRs may beidentified as the residues that make enthalpic contributions to antigenbinding. See, e.g., Makabe et al., Journal of Biological Chemistry,283:1156-1166, 2008. Still other CDR boundary definitions may notstrictly follow one of the above approaches, but will nonethelessoverlap with at least a portion of the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. As used herein, a CDR mayrefer to CDRs defined by any approach known in the art, includingcombinations of approaches. The methods used herein may utilize CDRsdefined according to any of these approaches. For any given embodimentcontaining more than one CDR, the CDRs may be defined in accordance withany of Kabat, Chothia, extended, AbM, contact, and/or conformationaldefinitions.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, Nature 256:495, 1975, ormay be made by recombinant DNA methods such as described in U.S. Pat.No. 4,816,567. The monoclonal antibodies may also be isolated from phagelibraries generated using the techniques described in McCafferty et al.,Nature 348:552-554, 1990, for example.

As used herein, “humanized” antibody refers to forms of non-human (e.g.murine) antibodies that are chimeric immunoglobulins, immunoglobulinchains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen binding subsequences of antibodies) that contain minimalsequence derived from non-human immunoglobulin. Preferably, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementarity determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Preferred areantibodies having Fc regions modified as described in WO 99/58572. Otherforms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDRL3, CDR H1, CDR H2, or CDR H3) which are altered with respect to theoriginal antibody, which are also termed one or more CDRs “derived from”one or more CDRs from the original antibody.

As used herein, “human antibody” means an antibody having an amino acidsequence corresponding to that of an antibody produced by a human and/orwhich has been made using any of the techniques for making humanantibodies known to those skilled in the art or disclosed herein. Thisdefinition of a human antibody includes antibodies comprising at leastone human heavy chain polypeptide or at least one human light chainpolypeptide. One such example is an antibody comprising murine lightchain and human heavy chain polypeptides. Human antibodies can beproduced using various techniques known in the art. In one embodiment,the human antibody is selected from a phage library, where that phagelibrary expresses human antibodies (Vaughan et al., NatureBiotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci.(USA) 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381,1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies canalso be made by immunization of animals into which human immunoglobulinloci have been transgenically introduced in place of the endogenousloci, e.g., mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016. Alternatively, the human antibody may be prepared byimmortalizing human B lymphocytes that produce an antibody directedagainst a target antigen (such B lymphocytes may be recovered from anindividual or from single cell cloning of the cDNA, or may have beenimmunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol.,147 (1):86-95, 1991; and U.S. Pat. No. 5,750,373.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to chains of amino acids of anylength, preferably, relatively short (e.g., 10-100 amino acids). Thechain may be linear or branched, it may comprise modified amino acids,and/or may be interrupted by non-amino acids. The terms also encompassan amino acid chain that has been modified naturally or by intervention;for example, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art. It is understood thatthe polypeptides can occur as single chains or associated chains.

A “monovalent antibody” comprises one antigen binding site per molecule(e.g., IgG or Fab). In some instances, a monovalent antibody can havemore than one antigen binding sites, but the binding sites are fromdifferent antigens.

A “monospecific antibody” comprises two identical antigen binding sitesper molecule (e.g. IgG) such that the two binding sites bind identicalepitope on the antigen. Thus, they compete with each other on binding toone antigen molecule. Most antibodies found in nature are monospecific.In some instances, a monospecific antibody can also be a monovalentantibody (e.g. Fab)

A “bivalent antibody” comprises two antigen binding sites per molecule(e.g., IgG). In some instances, the two binding sites have the sameantigen specificities. However, bivalent antibodies may be bispecific.

A “bispecific” or “dual-specific” is a hybrid antibody having twodifferent antigen binding sites. The two antigen binding sites of abispecific antibody bind to two different epitopes, which may reside onthe same or different protein targets.

A “bifunctional” is antibody is an antibody having identical antigenbinding sites (i.e., identical amino acid sequences) in the two arms buteach binding site can recognize two different antigens.

A “heteromultimer”, “heteromultimeric complex”, or “heteromultimericpolypeptide” is a molecule comprising at least a first polypeptide and asecond polypeptide, wherein the second polypeptide differs in amino acidsequence from the first polypeptide by at least one amino acid residue.The heteromultimer can comprise a “heterodimer” formed by the first andsecond polypeptide or can form higher order tertiary structures wherepolypeptides in addition to the first and second polypeptide arepresent.

A “heterodimer,” “heterodimeric protein,” “heterodimeric complex,” or“heteromultimeric polypeptide” is a molecule comprising a firstpolypeptide and a second polypeptide, wherein the second polypeptidediffers in amino acid sequence from the first polypeptide by at leastone amino acid residue.

The “hinge region,” “hinge sequence”, and variations thereof, as usedherein, includes the meaning known in the art, which is illustrated in,for example, Janeway et al., ImmunoBiology: the immune system in healthand disease, (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al.,Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods(1997), 209:193-202.

The “immunoglobulin-like hinge region,” “immunoglobulin-like hingesequence,” and variations thereof, as used herein, refer to the hingeregion and hinge sequence of an immunoglobulin-like or an antibody-likemolecule (e.g., immunoadhesins). In some embodiments, theimmunoglobulin-like hinge region can be from or derived from any IgG1,IgG2, IgG3, or IgG4 subtype, or from IgA, IgE, IgD or IgM, includingchimeric forms thereof, e.g., a chimeric IgG1/2 hinge region.

The term “immune effector cell” or “effector cell as used herein refersto a cell within the natural repertoire of cells in the human immunesystem which can be activated to affect the viability of a target cell.The viability of a target cell can include cell survival, proliferation,and/or ability to interact with other cells.

Antibodies of the invention can be produced using techniques well knownin the art, e.g., recombinant technologies, phage display technologies,synthetic technologies or combinations of such technologies or othertechnologies readily known in the art (see, for example, Jayasena, S.D., Clin. Chem., 45: 1628-50, 1999 and Fellouse, F. A., et al, J. Mol.Biol., 373(4):924-40, 2007).

As known in the art, “polynucleotide,” or “nucleic acid,” as usedinterchangeably herein, refer to chains of nucleotides of any length,and include DNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a chain by DNA or RNApolymerase. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thechain. The sequence of nucleotides may be interrupted by non-nucleotidecomponents. A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component. Othertypes of modifications include, for example, “caps”, substitution of oneor more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid supports. The 5′ and 3′ terminal OH can be phosphorylated orsubstituted with amines or organic capping group moieties of from 1 to20 carbon atoms. Other hydroxyls may also be derivatized to standardprotecting groups. Polynucleotides can also contain analogous forms ofribose or deoxyribose sugars that are generally known in the art,including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or2′-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomericsugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, sedoheptuloses, acyclic analogs and abasicnucleoside analogs such as methyl riboside. One or more phosphodiesterlinkages may be replaced by alternative linking groups. Thesealternative linking groups include, but are not limited to, embodimentswherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”),(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂ (“formacetal”), in whicheach R or R′ is independently H or substituted or unsubstituted alkyl(1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl,cycloalkyl, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. The preceding description applies toall polynucleotides referred to herein, including RNA and DNA.

As known in the art a “constant region” of an antibody refers to theconstant region of the antibody light chain or the constant region ofthe antibody heavy chain, either alone or in combination.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably, at least90% pure, more preferably, at least 95% pure, yet more preferably, atleast 98% pure, and most preferably, at least 99% pure.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

As known in the art, the term “Fc region” is used to define a C-terminalregion of an immunoglobulin heavy chain. The “Fc region” may be a nativesequence Fc region or a variant Fc region. Although the boundaries ofthe Fc region of an immunoglobulin heavy chain might vary, the human IgGheavy chain Fc region is usually defined to stretch from an amino acidresidue at position Cys226, or from Pro230, to the carboxyl-terminusthereof. The numbering of the residues in the Fc region is that of theEU index as in Kabat. Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991. The Fc region of animmunoglobulin generally comprises two constant regions, CH2 and CH3.

As used in the art, “Fc receptor” and “FcR” describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet,Ann. Rev. Immunol., 9:457-92, 1991; Capel et al., Immunomethods,4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995.“FcR” also includes the neonatal receptor, FcRn, which is responsiblefor the transfer of maternal IgGs to the fetus (Guyer et al., J.Immunol., 117:587, 1976; and Kim et al., J. Immunol., 24:249, 1994).

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or an antigen binding fragment (or portion)thereof, binds to an epitope in a manner sufficiently similar to thebinding of a second antibody, or an antigen binding portion thereof,such that the result of binding of the first antibody with its cognateepitope is detectably decreased in the presence of the second antibodycompared to the binding of the first antibody in the absence of thesecond antibody. The alternative, where the binding of the secondantibody to its epitope is also detectably decreased in the presence ofthe first antibody, can, but need not be the case. That is, a firstantibody can inhibit the binding of a second antibody to its epitopewithout that second antibody inhibiting the binding of the firstantibody to its respective epitope. However, where each antibodydetectably inhibits the binding of the other antibody with its cognateepitope or ligand, whether to the same, greater, or lesser extent, theantibodies are said to “cross-compete” with each other for binding oftheir respective epitope(s). Both competing and cross-competingantibodies are encompassed by the present invention. Regardless of themechanism by which such competition or cross-competition occurs (e.g.,steric hindrance, conformational change, or binding to a common epitope,or portion thereof), the skilled artisan would appreciate, based uponthe teachings provided herein, that such competing and/orcross-competing antibodies are encompassed and can be useful for themethods disclosed herein.

A “functional Fc region” possesses at least one effector function of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity; phagocytosis;down-regulation of cell surface receptors (e.g. B cell receptor), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays known in the art for evaluating suchantibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification, yet retains at least one effector function of the nativesequence Fc region. In some embodiments, the variant Fc region has atleast one amino acid substitution compared to a native sequence Fcregion or to the Fc region of a parent polypeptide, e.g. from about oneto about ten amino acid substitutions, and preferably, from about one toabout five amino acid substitutions in a native sequence Fc region or inthe Fc region of the parent polypeptide. The variant Fc region hereinwill preferably possess at least about 80% sequence identity with anative sequence Fc region and/or with an Fc region of a parentpolypeptide, and most preferably, at least about 90% sequence identitytherewith, more preferably, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99% sequenceidentity therewith.

The term “effector function” refers to the biological activitiesattributable to the Fc region of an antibody. Examples of antibodyeffector functions include, but are not limited to, antibody-dependentcell-mediated cytotoxicity (ADCC), Fc receptor binding, complementdependent cytotoxicity (CDC), phagocytosis, C1q binding, and downregulation of cell surface receptors (e.g., B cell receptor; BCR). See,e.g., U.S. Pat. No. 6,737,056. Such effector functions generally requirethe Fc region to be combined with a binding domain (e.g., an antibodyvariable domain) and can be assessed using various assays known in theart for evaluating such antibody effector functions. An exemplarymeasurement of effector function is through Fcγ3 and/or C1 q binding.

As used herein “antibody-dependent cell-mediated cytotoxicity” or “ADCC”refers to a cell-mediated reaction in which nonspecific cytotoxic cellsthat express Fc receptors (FcRs) (e.g. natural killer (NK) cells,neutrophils, and macrophages) recognize bound antibody on a target celland subsequently cause lysis of the target cell. ADCC activity of amolecule of interest can be assessed using an in vitro ADCC assay, suchas that described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and NK cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in an animalmodel such as that disclosed in Clynes et al., 1998, PNAS (USA),95:652-656.

“Complement dependent cytotoxicity” or “CDC” refers to the lysing of atarget in the presence of complement. The complement activation pathwayis initiated by the binding of the first component of the complementsystem (C1q) to a molecule (e.g. an antibody) complexed with a cognateantigen. To assess complement activation, a CDC assay, e.g. as describedin Gazzano-Santoro et al., J. Immunol. Methods, 202: 163 (1996), may beperformed.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: reducing the proliferation of (or destroying) neoplasticor cancerous cells, inhibiting metastasis of neoplastic cells, remissionof a BCMA associated disease (e.g., cancer or autoimmune disease),decreasing symptoms resulting from a BCMA associated disease (e.g.,cancer or autoimmune disease), increasing the quality of life of thosesuffering from a BCMA associated disease (e.g., cancer or autoimmunedisease), decreasing the dose of other medications required to treat aBCMA associated disease (e.g., cancer or autoimmune disease), delayingthe progression of a BCMA associated disease (e.g., cancer or autoimmunedisease), curing a BCMA associated disease (e.g., cancer or autoimmunedisease), and/or prolong survival of patients having a BCMA associateddisease (e.g., cancer or autoimmune disease).

“Ameliorating” means a lessening or improvement of one or more symptomsas compared to not administering a BCMA antibody or a BCMA antibodyconjugate. “Ameliorating” also includes shortening or reduction induration of a symptom.

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toeffect any one or more beneficial or desired results. For prophylacticuse, beneficial or desired results include eliminating or reducing therisk, lessening the severity, or delaying the outset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such as reducingincidence or amelioration of one or more symptoms of various BCMAassociated diseases or conditions (such as multiple myeloma), decreasingthe dose of other medications required to treat the disease, enhancingthe effect of another medication, and/or delaying the progression of theBCMA associated disease of patients. An effective dosage can beadministered in one or more administrations. For purposes of thisinvention, an effective dosage of drug, compound, or pharmaceuticalcomposition is an amount sufficient to accomplish prophylactic ortherapeutic treatment either directly or indirectly. As is understood inthe clinical context, an effective dosage of a drug, compound, orpharmaceutical composition may or may not be achieved in conjunctionwith another drug, compound, or pharmaceutical composition. Thus, an“effective dosage” may be considered in the context of administering oneor more therapeutic agents, and a single agent may be considered to begiven in an effective amount if, in conjunction with one or more otheragents, a desirable result may be or is achieved.

An “individual” or a “subject” is a mammal, more preferably, a human.Mammals also include, but are not limited to, farm animals, sportanimals, pets, primates, horses, dogs, cats, mice and rats.

As used herein, “vector” means a construct, which is capable ofdelivering, and, preferably, expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

As used herein, “pharmaceutically acceptable carrier” or “pharmaceuticalacceptable excipient” includes any material which, when combined with anactive ingredient, allows the ingredient to retain biological activityand is non-reactive with the subject's immune system. Examples include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution, water, emulsions such asoil/water emulsion, and various types of wetting agents. Preferreddiluents for aerosol or parenteral administration are phosphate bufferedsaline (PBS) or normal (0.9%) saline. Compositions comprising suchcarriers are formulated by well known conventional methods (see, forexample, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro,ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Scienceand Practice of Pharmacy 21st Ed. Mack Publishing, 2005).

The term “acyl donor glutamine-containing tag” or “glutamine tag” asused herein refers to a polypeptide or a protein containing one or moreGln residue(s) that acts as a transglutaminase amine acceptor. See,e.g., WO2012059882 and WO2015015448.

The term “k_(on)” or “k_(a)”, as used herein, refers to the rateconstant for association of an antibody to an antigen. Specifically, therate constants (k_(on)/k_(a) and k_(off)/k_(d)) and equilibriumdissociation constants are measured using whole antibody (i.e. bivalent)and monomeric BCMA proteins.

The term “k_(off)” or “k_(d)”, as used herein, refers to the rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(D)”, as used herein, refers to the equilibrium dissociationconstant of an antibody-antigen interaction.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent specification, including definitions, will control. Throughoutthis specification and claims, the word “comprise,” or variations suchas “comprises” or “comprising” will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers. Unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular.

Exemplary methods and materials are described herein, although methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the present invention. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BCMA Antibodies and Methods of Making Thereof

The present invention provides an antibody that binds to BCMA (e.g.,human BCMA (e.g., SEQ ID NO: 353 or accession number: Q02223-2) andcharacterized by any one or more of the following characteristics: (a)treat, prevent, ameliorate one or more symptoms of a conditionassociated with malignant cells expressing BCMA in a subject (e.g.,B-cell related cancer such as multiple myeloma); (b) inhibit tumorgrowth or progression in a subject (who has a malignant tumor expressingBCMA); (c) inhibit metastasis of cancer (malignant) cells expressingBCMA in a subject (who has one or more malignant cells expressing BCMA);(f) induce regression (e.g., long-term regression) of a tumor expressingBCMA; (d) exert cytotoxic activity in malignant cells expressing BCMA;and (e) block BCMA interaction with other yet to be identified factors.

In one aspect, provided is an isolated antibody, or an antigen bindingfragment thereof, which specifically binds to B-Cell Maturation Antigen(BCMA), wherein the antibody comprises (a) a heavy chain variable (VH)region comprising (i) a VH complementary determining region one (CDR1)comprising the sequence SYX₁MX₂, wherein X₁ is A or P; and X₂ is T, N,or S (SEQ ID NO: 301), GFTFX₁SY, wherein X₁ is G or S (SEQ ID NO: 302),or GFTFX₁SYX₂MX₃, wherein X₁ is G or S, X₂ is A or P; and X₃ is T, N, orS (SEQ ID NO: 303); (ii) a VH CDR2 comprising the sequenceAX₁X₂X₃X₄GX₅X₆X₇X₈YADX₉X₁₀KG, wherein X₁ is I, V, T, H, L, A, or C; X₂is S, D, G, T, I, L, F, M, or V; X₃ is G, Y, L, H, D, A, S, or M; X₄ is5, Q, T, A, F, or W; X₅ is G or T; X₆ is N, S, P, Y, W, or F; X₇ is S,T, I, L, T, A, R, V, K, G, or C; X₈ is F, Y, P, W, H, or G; X₉ is V, R,or L; and X₁₀ is G or T (SEQ ID NO: 305), or X₁X₂X₃X₄X₅X₆, wherein X₁ isS, V, D, G, T, L, F, or M; X₂ is G, Y, L, H, D, A, S, or M; X₃ is S, G,F, or W; X₄ is G or S; X₅ is G or T; and X₆ is N, S, P, Y, or W (SEQ IDNO: 306); and iii) a VH CDR3 comprising the sequence VSPIX₁X₂X₃X₄,wherein X₁ is A or Y; X₂ is A or S; and X₃ is G, Q, L, P, or E (SEQ IDNO: 307), or YWPMX₁X₂, wherein X₁ is D, S, T, or A; and X₂ is I, S, L,P, or D (SEQ ID NO: 308); and/or a light chain variable (VL) regioncomprising (i) a VL CDR1 comprising the sequenceX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂, wherein X₁ is R, G, W, A, or C; X₂ is A, P,G, L, C, or S; X₃ is S, G, or R; X₄ is Q, C, E, V, or I; X₅ is S, P, G,A, R, or D; X₆ is V, G, I, or L; X₇ is S, E, D, P, or G; X₈ is S, P, F,A, M, E, V, N, D, or Y; X₉ is T, V, E, S, A, M, Q, Y, H, R, or F; X₁₀ isY or F; X₁₁ is L, W, or P; and X₁₂ is A, S, or G (SEQ ID NO: 309); (ii)a VL CDR2 comprising the sequence X₁ASX₂RAX₃, wherein X₁ is G or D; X₂is S or I; and X₃ is T or P (SEQ ID NO: 310); and (iii) a VL CDR3comprising the sequence QQYX₁X₂X₃PX₄T, wherein X₁ is G, Q, E, L, F, A,S, M, K, R, or Y; X₂ is S, R, T, G, V, F, Y, D, A, H, V, E, K, or C; X₃is W, F, or S; and X₄ is L or I (SEQ ID NO: 311), or QQYX₁X₂X₃PX₄,wherein X₁ is G, Q, E, L, F, A, S, M, R, K, or Y; X₂ is S, R, T, G, R,V, D, A, H, E, K, C, F, or Y; X₃ is W, 5, or F; and X₄ is L or I (SEQ IDNO: 312).

In another aspect, provided is an isolated antibody, or an antigenbinding fragment thereof, which specifically binds to BCMA, wherein theantibody comprises: a VH region comprising a VH CDR1, VH CDR2, and VHCDR3 of the VH sequence shown in SEQ ID NO: 2, 3, 7, 8, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 35, 37, 39, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 83, 87, 92, 95, 97, 99, 101,104, 106, 110, 112, 114, 118, 120, 122, 112, 125, 127, 313, 314, 363, or365; and/or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of theVL sequence shown in SEQ ID NO: 1, 4, 5, 6, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 317, 80, 81, 82,84, 85, 86, 88, 89, 90, 91, 93, 94, 96, 98, 100, 102, 103, 105, 107,108, 109, 111, 113, 115, 116, 117, 119, 121, 123, 124, 126, 128, 315,316, or 364.

In some embodiments, provided is an antibody having any one of partiallight chain sequence as listed in Table 1 and/or any one of partialheavy chain sequence as listed in Table 1.

TABLE 1 mAb Light Chain Heavy Chain P6E01/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA P6E01 ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YGSPPSFT FGQGTKVEIK(SEQ YYCAR VSPIASGMDY WGQGTLVT ID NO: 1) VSS (SEQ ID NO: 2) P6E01/EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA H3.AQ ASQSVSSSYLAWYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YGSPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAQMDYWGQGTLVT ID NO: 1) VSS (SEQ ID NO: 3) L1.LGF/ EIVLTQSPGTLSLSPGERATLSCEVQLLESGGGLVQPGGSLRLSCA L3.KW/ RASQSLGSFYLA WYQQKPGQA ASGFTFGSYAMTWVRQAPGKGLE P6E01 PRLLIY GASSRAT GIPDRFSGSGWVSAISGSGGNTFYADSVKGRFTI SGTDFTLTISRLEPEDFAVYYC KHSRDNSKNTLYLQMNSLRAEDTAV YGWPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 4) VSS (SEQ ID NO: 2) L1.LGF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.NY/ ASQSLGSFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE P6E01 RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YNYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 5) VSS (SEQ ID NO: 2) L1.GDF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.NY/ ASQSVGDFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE P6E01 RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YNYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 6) VSS (SEQ ID NO: 2) L1.LGF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KW/ ASQSLGSFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AL RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KHYSRDNSKNTLYLQMNSLRAEDTAV GWPPSFT FGQGTKVEIK (SEQ ID YYCAR ARVSPIAALMDYWGQGTL NO: 4) VTVSS (SEQ ID NO: 7) L1.LGF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KW/ ASQSLGSFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AP RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KHYSRDNSKNTLYLQMNSLRAEDTAV GWPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIAAPMDYWGQGTLVT NO: 4) VSS (SEQ ID NO: 8) L1.LGF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KW/ ASQSLGSFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQ RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KHYSRDNSKNTLYLQMNSLRAEDTAV GWPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIAAQMDYWGQGTLVT NO: 4) VSS (SEQ ID NO: 3) L1.LGF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.PY/ ASQSLGSFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AP RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAPMDYWGQGTLVT ID NO: 9) VSS (SEQ ID NO: 8) L1.LGF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.PY/ ASQSLGSFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQ RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAQMDYWGQGTLVT ID NO: 9) VSS (SEQ ID NO: 3) L1.LGF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.NY/ ASQSLGSFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AL RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YNYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAALMDYWGQGTLVT ID NO: 10) VSS (SEQ ID NO: 7) L1.LGF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.NY/ ASQSLGSFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AP RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YNYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAPMDYWGQGTLVT ID NO: 10) VSS (SEQ ID NO: 8) L1.LGF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.NY/ ASQSLGSFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQ RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YNYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAQMDYWGQGTLVT ID NO: 10) VSS (SEQ ID NO: 3) L1.GDF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KW/ ASQSVGDFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AL RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KHYSRDNSKNTLYLQMNSLRAEDTAV GWPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIAALMDYWGQGTLVT NO: 11) VSS (SEQ ID NO: 7) L1.GDF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KW/ ASQSVGDFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AP RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KHYSRDNSKNTLYLQMNSLRAEDTAV GWPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIAAPMDYWGQGTLVT NO: 11) VSS (SEQ ID NO: 8) L1.GDF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KW/ ASQSVGDFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQ RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KHYSRDNSKNTLYLQMNSLRAEDTAV GWPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIAAQMDYWGQGTLVT NO: 11) VSS (SEQ ID NO: 3) L1.GDF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.PY/ ASQSVGDFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQ RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAQMDYWGQGTLVT ID NO: 12) VSS (SEQ ID NO: 3) L1.GDF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.NY/ ASQSVGDFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AL RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YNYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAALMDYWGQGTLVT ID NO: 13) VSS (SEQ ID NO: 7) L1.GDF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.NY/ ASQSVGDFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AP RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YNYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAPMDYWGQGTLVT ID NO: 13) VSS (SEQ ID NO: 8) L1.GDF/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.NY/ ASQSVGDFYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQ RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YNYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAQMDYWGQGTLVT ID NO: 14) VSS (SEQ ID NO: 3) L3.KW/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA P6E01 ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYC KHY SRDNSKNTLYLQMNSLRAEDTAV GWPPSFT FGQGTKVEIK(SEQ ID YYCAR VSPIASGMDY WGQGTLVT NO: 15) VSS (SEQ ID NO: 2) L3.PY/EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA P6E01 ASQSVSSSYLAWYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 16) VSS (SEQ ID NO: 2) L3.NY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA P6E01 ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YNYPPSFT FGQGTKVEIK(SEQ YYCAR VSPIASGMDY WGQGTLVT ID NO: 17) VSS (SEQ ID NO: 2) L3.PY/EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.PS/ ASQSVSSSYPSWYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE P6E01 RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 18) VSS (SEQ ID NO: 2) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.AH/ ASQSVSAHYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE P6E01 RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 19) VSS (SEQ ID NO: 2) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLA WYQQKPGQAPR ASGFTFGSYAMTWVRQAPGKGLE P6E01 LLIY GASSRAT GIPDRFSGSGSGWVSAISGSGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 20) VSS (SEQ ID NO: 2) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.PH/ ASQSVSPHYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE P6E01 RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 21) VSS (SEQ ID NO: 2) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KY/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE P6E01 RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KYYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 2) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KF/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE P6E01 RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KFYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 2) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA H2.QR ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGS WVSAISGSGGNTFYADQRKGRFTIGTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK(SEQ YYCAR VSPIASGMDY WGQGTLVT ID NO: 16) VSS (SEQ ID NO: 24) L3.PY/EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA H2.DY ASQSVSSSYLAWYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGSWVSAIDYSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 16) VSS (SEQ ID NO: 25) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA H2.YQ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGS WVSAISYQGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK(SEQ YYCAR VSPIASGMDY WGQGTLVT ID NO: 16) VSS (SEQ ID NO: 26) L3.PY/EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA H2.LT ASQSVSSSYLAWYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGSWVSAISLTGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 16) VSS (SEQ ID NO: 27) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA H2.HA ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGS WVSAISHAGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK(SEQ YYCAR VSPIASGMDY WGQGTLVT ID NO: 16) VSS (SEQ ID NO: 28) L3.PY/EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA H2.QL ASQSVSSSYLAWYQQKPGQAPRLLI ASGFTFG SYAMTWVRQAPGKGLE Y GASSRAT GIPDRFSGSGSGTDFTWVSAISGSGGNTFYADQLKGRFTI LTISRLEPEDFAVYYC QHYPYPPSFTSRDNSKNTLYLQMNSLRAEDTAV FGQGTKVEIK (SEQ ID NO: 16) YYCAR VSPIASGMDYWGQGTLVT VSS (SEQ ID NO: 29) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA H3.YA ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGS WVSAISGSGGNTFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK(SEQ YYCAR VSPIYAGMDY WGQGTLVT ID NO: 16) VSS (SEQ ID NO: 30) L3.PY/EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA H3.AE ASQSVSSSYLAWYQQKPGQAP ASGFTFG SY AMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGS WVSAISGSGGN TFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAEMDY WGQGTLVT ID NO: 16) VSS (SEQID NO: 31) L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAH3.AQ ASQSVSSSYLA WYQQKPGQAP ASGFTFG SY AMTWVRQAPGKGLE RLLIY GASSRATGIPDRFSGSGS WVSAI SGSGGN TFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAQMDYWGQGTLVT ID NO: 16) VSS (SEQ ID NO: 3) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA H3.TAQ ASQSVSSSYLA WYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGS WVSAI SGSGGN TFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK(SEQ YYCTR VSPIAAQMDY WGQGTLVT ID NO: 16) VSS (SEQ ID NO: 32) L3.PY/EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA P6E01 ASQSVSSSYLAWYQQKPGQAP ASGFTFG SY AMTWVRQAPGKGLE RLLIY GASSRAT GIPDRFSGSGS WVSAISGSGGN TFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAVYPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDY WGQGTLVT ID NO: 16) VSS (SEQID NO: 2) L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAL1.PS/ ASQSVSSSYPS WYQQKPGQAP ASGFTFG SY AMTWVRQAPGKGLE H2.QR RLLIYGASSRAT GIPDRFSGSGS WVSAI SGSGGN TFYADQRKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 18) VSS (SEQ ID NO: 24) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.PS/ ASQSVSSSYPS WYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE H2.DY RLLIY GASSRAT GIPDRFSGSGS WVSAI DYSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCAR VSPIASGMDY WGQGTLVT ID NO: 18) VSS (SEQ ID NO: 25)L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.PS/ASQSVSSSYPS WYQQKPGQAP ASGFTFG SY AMTWVRQAPGKGLE H2.YQ RLLIY GASSRATGIPDRFSGSGS WVSAI SYQGGN TFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK(SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 18) VSS (SEQ ID NO: 26) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.PS/ ASQSVSSSYPS WYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE H2.LT RLLIY GASSRAT GIPDRFSGSGS WVSAI SLTGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCAR VSPIASGMDY WGQGTLVT ID NO: 18) VSS (SEQ ID NO: 27)L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.PS/ASQSVSSSYPS WYQQKPGQAP ASGFTFG SY AMTWVRQAPGKGLE H2.HA RLLIY GASSRATGIPDRFSGSGS WVSAI SHAGGN TFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 18) VSS (SEQ ID NO: 28) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.PS/ ASQSVSSSYPS WYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE H2.QL RLLIY GASSRAT GIPDRFSGSGS WVSAI SGSGGNTFYADQLKGRFTI GTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCAR VSPIASGMDY WGQGTLVT ID NO: 18) VSS (SEQ ID NO: 29)L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.PS/ASQSVSSSYPS WYQQKPGQAP ASGFTFG SY AMTWVRQAPGKGLE H3.YA RLLIY GASSRATGIPDRFSGSGS WVSAI SGSGGN TFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIYAGMDYWGQGTLVT ID NO: 18) VSS (SEQ ID NO: 30) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.PS/ ASQSVSSSYPS WYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE H3.AE RLLIY GASSRAT GIPDRFSGSGS WVSAI SGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCAR VSPIAAEMDY WGQGTLVT ID NO: 18) VSS (SEQ ID NO: 31)L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.PS/ASQSVSSSYPS WYQQKPGQAP ASGFTFG SY AMTWVRQAPGKGLE H3.AQ RLLIY GASSRATGIPDRFSGSGS WVSAI SGSGGN TFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAQMDYWGQGTLVT ID NO: 18) VSS (SEQ ID NO: 3) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.PS/ ASQSVSSSYPS WYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE H3.TAQ RLLIY GASSRAT GIPDRFSGSGS WVSAI SGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCTR VSPIAAQMDY WGQGTLVT ID NO: 18) VSS (SEQ ID NO: 32)L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.AH/ASQSVSAHYLA WYQQKPGQAP ASGFTFG SY AMTWVRQAPGKGLE H2.QR RLLIY GASSRATGIPDRFSGSGS WVSAI SGSGGN TFYADQRKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 19) VSS (SEQ ID NO: 24) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.AH/ ASQSVSAHYLA WYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE H2.DY RLLIYGASSRATGIPDRFSGSGS WVSAI DYSGGN TFYADSVKGRFTIGTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQYYCAR VSPIASGMDY WGQGTLVT ID NO: 19) VSS (SEQ ID NO: 25) L3.PY/EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.AH/ ASQSVSAHYLAWYQQKPGQAP ASGFTFG SY AMTWVRQAPGKGLE H2.YQ RLLIY GASSRAT GIPDRFSGSGSWVSAI SYQGGN TFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 19) VSS (SEQ ID NO: 26) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.AH/ ASQSVSAHYLA WYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE H2.LT RLLIY GASSRAT GIPDRFSGSGS WVSAI SLTGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCAR VSPIASGMDY WGQGTLVT ID NO: 19) VSS (SEQ ID NO: 27)L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.AH/ASQSVSAHYLA WYQQKPGQAP ASGFTFG SY AMTWVRQAPGKGLE H2.HA RLLIY GASSRATGIPDRFSGSGS WVSAI SHAGGN TFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 19) VSS (SEQ ID NO: 28) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.AH/ ASQSVSAHYLA WYQQKPGQAP ASGFTFGS YAMTWVRQAPGKGLE H2.QL RLLIY GASSRAT GIPDRFSGSGS WVSAI SGSGGNTFYADQLKGRFTI GTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK(SEQ YYCAR VSPIASGMDY WGQGTLVT ID NO: 19) VSS (SEQ ID NO: 29)L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.AH/ASQSVSAHYLA WYQQKPGQAP ASGFTFG SY AMTWVRQAPGKGLE H3.YA RLLIY GASSRATGIPDRFSGSGS WVSAI SGSGGN TFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIYAGMDYWGQGTLVT ID NO: 19) VSS (SEQ ID NO: 30) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.AH/ ASQSVSAHYLA WYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE H3.AE RLLIY GASSRAT GIPDRFSGSGS WVSAI SGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCAR VSPIAAEMDY WGQGTLVT ID NO: 19) VSS (SEQ ID NO: 31)L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.AH/ASQSVSAHYLA WYQQKPGQAP ASGFTFG SY AMTWVRQAPGKGLE H3.AQ RLLIY GASSRATGIPDRFSGSGS WVSAI SGSGGN TFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAQMDYWGQGTLVT ID NO: 19) VSS (SEQ ID NO: 3) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.AH/ ASQSVSAHYLA WYQQKPGQAP ASGFTFG SYAMTWVRQAPGKGLE H3.TAQ RLLIY GASSRAT GIPDRFSGSGS WVSAI SGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QH SRDNSKNTLYLQMNSLRAEDTAV YPYPPSFTFGQGTKVEIK (SEQ YYCTR VSPIAAQMDY WGQGTLVT ID NO: 19) VSS (SEQ ID NO: 32)L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.FF/ASQSVSSFFLA WYQQKPGQAPR ASGFTFG SY AMTWVRQAPGKGLE H2.QR LLIY GASSRATGIPDRFSGSGSG WVSAI SGSGGN TFYADQRKGRFTI TDFTLTISRLEPEDFAVYYC QHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 20) VSS (SEQ ID NO: 24) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLA WYQQKPGQAPR ASGFTFG SYAMTWVRQAPGKGLE H2.DY LLIY GASSRAT GIPDRFSGSGSG WVSAI DYSGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QHYP SRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ ID YYCAR VSPIASGMDY WGQGTLVT NO: 20) VSS (SEQ ID NO: 25)L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.FF/ASQSVSSFFLA WYQQKPGQAPR ASGFTFG SY AMTWVRQAPGKGLE H2.YQ LLIY GASSRATGIPDRFSGSGSG WVSAI SYQGGN TFYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 20) VSS (SEQ ID NO: 26) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLA WYQQKPGQAPR ASGFTFG SYAMTWVRQAPGKGLE H2.LT LLIY GASSRAT GIPDRFSGSGSG WVSAI SLTGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QHYP SRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ ID YYCAR VSPIASGMDY WGQGTLVT NO: 20) VSS (SEQ ID NO: 27)L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.FF/ASQSVSSFFLA WYQQKPGQAPR ASGFTFG SY AMTWVRQAPGKGLE H2.HA LLIY GASSRATGIPDRFSGSGSG WVSAI SHAGGN TFYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 20) VSS (SEQ ID NO: 28) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLA WYQQKPGQAPR ASGFTFG SYAMTWVRQAPGKGLE H2.QL LLIY GASSRAT GIPDRFSGSGSG WVSAI SGSGGNTFYADQLKGRFTI TDFTLTISRLEPEDFAVYYC QHYP SRDNSKNTLYLQMNSLRAEDTAV YPPSFTFGQGTKVEIK (SEQ ID YYCAR VSPIASGMDY WGQGTLVT NO: 20) VSS (SEQ ID NO: 29)L3.PY/ EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA L1.FF/ASQSVSSFFLA WYQQKPGQAPR ASGFTFG SY AMTWVRQAPGKGLE H3.YA LLIY GASSRATGIPDRFSGSGSG WVSAI SGSGGN TFYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIYAGMDYWGQGTLVT NO: 20) VSS (SEQ ID NO: 30) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLA WYQQKPGQAPR ASGFTFGSYAMTWVRQAPGKGLE H3.AE LLIY GASSRAT GIPDRFSGSGSGWVSAISGSGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIAAEMDYWGQGTLVT NO: 20) VSS (SEQ ID NO: 31) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLA WYQQKPGQAPR ASGFTFGSYAMTWVRQAPGKGLE H3.AQ LLIY GASSRAT GIPDRFSGSGSGWVSAISGSGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIAAQMDYWGQGTLVT NO: 20) VSS (SEQ ID NO: 3) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.FF/ ASQSVSSFFLA WYQQKPGQAPR ASGFTFGSYAMTWVRQAPGKGLE H3.TAQ LLIY GASSRAT GIPDRFSGSGSGWVSAISGSGGNTFYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QHYPSRDNSKNTLYLQMNSLRAEDTAV YPPSFT FGQGTKVEIK (SEQ ID YYCTR VSPIAAQMDYWGQGTLVT NO: 20) VSS (SEQ ID NO: 32) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.PH/ ASQSVSPHYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.QR RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADQRKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 21) VSS (SEQ ID NO: 24) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.PH/ ASQSVSPHYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.HA RLLIY GASSRAT GIPDRFSGSGSWVSAISHAGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIASGMDYWGQGTLVT ID NO: 21) VSS (SEQ ID NO: 28) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.PH/ ASQSVSPHYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AE RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAEMDYWGQGTLVT ID NO: 21) VSS (SEQ ID NO: 31) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.PH/ ASQSVSPHYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQ RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCAR VSPIAAQMDYWGQGTLVT ID NO: 21) VSS (SEQ ID NO: 3) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L1.PH/ ASQSVSPHYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.TAQ RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YPYPPSFT FGQGTKVEIK (SEQ YYCTR VSPIAAQMDYWGQGTLVT ID NO: 21) VSS (SEQ ID NO: 32) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KY/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.QR RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADQRKGRFTI GTDFTLTISRLEPEDFAVYYC KYYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 24) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KY/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.DY RLLIY GASSRAT GIPDRFSGSGSWVSAIDYSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KYYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 25) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KY/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.YQ RLLIY GASSRAT GIPDRFSGSGSWVSAISYQGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KYYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 26) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KY/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.LT RLLIY GASSRAT GIPDRFSGSGSWVSAISLTGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KYYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 27) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KY/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.HA RLLIY GASSRAT GIPDRFSGSGSWVSAISHAGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KYYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 28) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KY/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.QL RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADQLKGRFTI GTDFTLTISRLEPEDFAVYYC KYYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 29) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KY/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.YA RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KYYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIYAGMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 30) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KY/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.TAQ RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KYYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCTR VSPIAAQMDYWGQGTLVT NO: 22) VSS (SEQ ID NO: 32) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KF/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.DY RLLIY GASSRAT GIPDRFSGSGSWVSAIDYSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KFYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 25) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KF/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.YQ RLLIY GASSRAT GIPDRFSGSGSWVSAISYQGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KFYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 26) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KF/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.LT RLLIY GASSRAT GIPDRFSGSGSWVSAISLTGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KFYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 27) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KF/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H2.QL RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADQLKGRFTI GTDFTLTISRLEPEDFAVYYC KFYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIASGMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 29) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KF/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.YA RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KFYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIYAGMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 30) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KF/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AE RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KFYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIAAEMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 31) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KF/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.AQ RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KFYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCAR VSPIAAQMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 3) L3.PY/ EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA L3.KF/ ASQSVSSSYLA WYQQKPGQAP ASGFTFGSYAMTWVRQAPGKGLE H3.TAQ RLLIY GASSRAT GIPDRFSGSGSWVSAISGSGGNTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC KFYSRDNSKNTLYLQMNSLRAEDTAV PYPPSFT FGQGTKVEIK (SEQ ID YYCTR VSPIAAQMDYWGQGTLVT NO: 23) VSS (SEQ ID NO: 32) P5A2_VHVL EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYLA WYQQKPGQAP ASGFTFS SYAMNWVRQAPGKGLERLLMY DASIRAT GIPDRFSGSGS WVSAISDSGGS TYYADSVKGRFTIGTDFTLTISRLEPEDFAVYYC QQ SRDNSKNTLYLQMNSLRAEDTAV YGSWPLT FGQGTKVEIK (SEQID YYCAR YWPMDI WGQGTLVTVSS NO: 34) (SEQ ID NO: 33) A02_Rd4_0.6nM_C06EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSVIYLAWYQQKPGQAPR ASGFTFS SYAMNWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSG WVSAISDSGGS AWYADSVKGRFT TDFTLTISRLEPEDFAVYYC QQY ISRDNSKNTLYLQMNSLRAEDTAVQRWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMSL WGQGTLVTVSS NO: 36) (SEQ ID NO:35) A02_Rd4_0.6nM_C09 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSSSYLA WYQQKPGQAP ASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRATGIPDRFSGSGS WVSAI SDSGGS MWYADSVKGRF GTDFTLTISRLEPEDFAVYYC QQTISRDNSKNTLYLQMNSLRAEDTA YQSWPLT FGQGTKVEIK (SEQ ID VYYCAR YWPMSLWGQGTLVTVS NO: 38) S (SEQ ID NO: 37) A02_Rd4_6nM_C16EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSDIYLAWYQQKPGQAPR ASGFTFS SYAMNWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSG WVSAISdFGGS TYYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQY SRDNSKNTLYLQMNSLRAEDTAVQTWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDI WGQGTLVTVSS NO: 40) (SEQ ID NO:39) A02_Rd4_6nM_C03 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSNLYLA WYQQKPGQAP ASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRATGIPDRFSGSGS WVSAISDSGGS TYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QQSRDNSKNTLYLQMNSLRAEDTAV YQGWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDIWGQGTLVTVSS NO: 41) (SEQ ID NO: 33) A02_Rd4_6nM_C01EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSAYYLA WYQQKPGQAPASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRAT GIPDRFSGSGS WVSAI TASGGSTYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QQ SRDNSKNTLYLQMNSLRAEDTAV YERWPLTFGQGTKVEIK (SEQ ID YYCAR YWPMSL WGQGTLVTVSS NO: 43) (SEQ ID NO: 42)A02_Rd4_6nM_C26 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSSLYLA WYQQKPGQAPR ASGFTFS SYAMNWVRQAPGKGLE LLMY DASIRATGIPDRFSGSGSG WVSAISDSGGS TYYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQYSRDNSKNTLYLQMNSLRAEDTAV QVWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMSLWGQGTLVTVSS NO: 45) (SEQ ID NO: 44) A02_Rd4_6nM_C25EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYLA WYQQKPGQAPASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRAT GIPDRFSGSGS WVSAI SdSGGSRWYADSVKGRFT GTDFTLTISRLEPEDFAVYYC QQ ISRDNSKNTLYLQMNSLRAEDTAV YLDWPLTFGQGTKVEIK (SEQ ID YYCAR YWPMTP WGQGTLVTVSS NO: 47) (SEQ ID NO: 46)A02_Rd4_6nM_C22 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSSSYLA WYQQKPGQAP ASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRATGIPDRFSGSGS WVSAVLdSGGSTYYADSVKGRFT GTDFTLTISRLEPEDFAVYYC QQISRDNSKNTLYLQMNSLRAEDTAV YQVWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMTPWGQGTLVTVSS NO: 49) (SEQ ID NO: 48) A02_Rd4_6nM_C19EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSVIYLAWYQQKPGQAPR ASGFTFS SYAMNWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSG WVSAISdSGGS RWYADSVKGRFT TDFTLTISRLEPEDFAVYYC QQYL ISRDNSKNTLYLQMNSLRAEDTAVAWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMSD WGQGTLVTVSS NO: 51) (SEQ ID NO:50) A02_Rd4_0.6nM_C03 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSSSYLA WYQQKPGQAP ASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRATGIPDRFSGSGS WVSAI SdSGG SKWYADSVKGRFT GTDFTLTISRLEPEDFAVYYC QQISRDNSKNTLYLQMNSLRAEDTAV YFTWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMSLWGQGTLVTVSS NO: 53) (SEQ ID NO: 52) A02_Rd4_6nM_C07EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSPvYLAWYQQKPGQAPR ASGFTFS SYAMNWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSG WVSAIGgSGGS LPYADSVKGRFT TDFTLTISRLEPEDFAVYYC QQYE ISRDNSKNTLYLQMNSLRAEDTAVRWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDS WGQGTLVTVSS NO: 55) (SEQ ID NO:54) A02_Rd4_6nM_C23 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSVEYLA WYQQKPGQAP ASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRATGIPDRFSGSGS WVSAI SdSGGS GWYADSVKGRFT GTDFTLTISRLEPEDFAVYYC QQISRDNSKNTLYLQMNSLRAEDTAV YARWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMSLWGQGTLVTVSS NO: 57) (SEQ ID NO: 56) A02_Rd4_0.6nM_C18EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSEIYLAWYQQKPGQAPR ASGFTFS SYAMNWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSGWVSAVLdSGGSTYYADSVKGRFT TDFTLTISRLEPEDFAVYYC QQYFISRDNSKNTLYLQMNSLRAEDTAV GWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMSLWGQGTLVTVSS NO: 59) (SEQ ID NO: 58) A02_Rd4_6nM_C10EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVEMSYLA WYQQKPGQAPASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRAT GIPDRFSGSGS WVSAI SdSGGSCWYADSVKGRFT GTDFTLTISRLEPEDFAVYYC QQ ISRDNSKNTLYLQMNSLRAEDTAV YAHWPLTFGQGTKVEIK (SEQ ID YYCAR YWPMTP WGQGTLVTVSS NO: 61) (SEQ ID NO: 60)A02_Rd4_6nM_C05 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSSSYLA WYQQKPGQAP ASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRATGIPDRFSGSGS WVSAIFaSGGSTYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QQSRDNSKNTLYLQMNSLRAEDTAV YQRWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMTPWGQGTLVTVSS NO: 63) (SEQ ID NO: 62) A02_Rd4_0.6nM_C10EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSAQYLA WYQQKPGQAPASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRAT GIPDRFSGSGSWVSAISgWGGSLPYADSVKGRFT GTDFTLTISRLEPEDFAVYYC QQISRDNSKNTLYLQMNSLRAEDTAV YQRWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDSWGQGTLVTVSS NO: 65) (SEQ ID NO: 64) A02_Rd4_6nM_C04EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSAIYLAWYQQKPGQAPR ASGFTFS SYAMNWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSGWVSAIMsSGGPLYYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQYSRDNSKNTLYLQMNSLRAEDTAV QVWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMALWGQGTLVTVSS NO: 67) (SEQ ID NO: 66) A02_Rd4_0.6nM_C26EIVLTQSPGTLSLSPGERATLSC G EVQLLESGGGLVQPGGSLRLSCA PSQSVSSSYLAWYQQKPGQAPR ASGFTFS SYAMNWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSGWVSAILmSGGSTYYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQYSRDNSKNTLYLQMNSLRAEDTAV QSWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMSLWGQGTLVTVSS NO: 69) (SEQ ID NO: 68) A02_Rd4_0.6nM_C13EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYWA WYQQKPGQAPASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRAT GIPDRFSGSGSWVSAISdSGGYRYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QQSRDNSKNTLYLQMNSLRAEDTAV YESWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMSLWGQGTLVTVSS NO: 71) (SEQ ID NO: 70) A02_Rd4_0.6nM_C01EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA GGQSVSSSYLA WYQQKPGQAPASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRAT GIPDRFSGSGSWVSAILsSGGSTYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QQSRDNSKNTLYLQMNSLRAEDTAV YQSWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDIWGQGTLVTVSS NO: 73) (SEQ ID NO: 72) A02_Rd4_6nM_C08EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSFIYLAWYQQKPGQAPR ASGFTFS SYAMNWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSGWVSAILdSGGSTYYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQYSRDNSKNTLYLQMNSLRAEDTAV GSWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMSPWGQGTLVTVSS NO: 75) (SEQ ID NO: 74) P5C1_VHVL EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA ASQSVSSTYLA WYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLELLIY DASSRAP GIPDRFSGSGSG WVSAI GgSGGS TYYADSVKGRFT TDFTLTISRLEPEDFAVYYCQQYS ISRDNSKNTLYLQMNSLRAEDTAV TSPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDSWGQGTLVTVSS NO: 77) (SEQ ID NO: 76) C01_Rd4_6nM_C24EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSPEYLA WYQQKPGQAPASGFTFS SYPMSWVRQAPGKGLE RLLIY DASSRAP GIPDRFSGSGS WVSAI GgSGGSLPYADSVKGRFT GTDFTLTISRLEPEDFAVYYC QQ ISRDNSKNTLYLQMNSLRAEDTAV YSVWPLTFGQGTKVEIK (SEQ ID YYCAR YWPMDS WGQGTLVTVSS NO: 79) (SEQ ID NO: 78)C01_Rd4_6nM_C26 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSAIYLA WYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLIY DASSRAPGIPDRFSGSGSG WVSAI GgSGGS LPYADSVKGRFT TDFTLTISRLEPEDFAVYYCQ QYSISRDNSKNTLYLQMNSLRAEDTAV AWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDSWGQGTLVTVSS NO: 317) (SEQ ID NO: 78) C01_Rd4_6nM_C10EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSSvYLAWYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLIY DASSRAP GIPDRFSGSGSG WVSAIGgSGGS LPYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQYS SRDNSKNTLYLQMNSLRAEDTAVTWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDS WGQGTLVTVSS NO: 79) (SEQ ID NO:78) C01_Rd4_0.6nM_C27 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSSTYLA WYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLIY DASSRAPGIPDRFSGSGSG WVSAI GgSGGS LPYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQYSSRDNSKNTLYLQMNSLRAEDTAV RWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDSWGQGTLVTVSS NO: 81) (SEQ ID NO: 78) C01_Rd4_6nM_C20EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSPIYLAWYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLIY DASSRAP GIPDRFSGSGSG WVSAIGgSGGS LPYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQYS SRDNSKNTLYLQMNSLRAEDTAVAFPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDS WGQGTLVTVSS NO: 82) (SEQ ID NO:78) C01_Rd4_6nM_C12 EIVLTQSPGTLSLSPGERATLSC EVQLLESGGGLVQPGGSLRLSCAWLSQSVSSTYLA WYQQKPGQA ASGFTFS SYPMSWVRQAPGKGLE PRLLIY DASSRAPGIPDRFSGSG WVSAIGgSGGWSYYADSVKGRFT SGTDFTLTISRLEPEDFAVYYC QISRDNSKNTLYLQMNSLRAEDTAV QYSEWPLT FGQGTKVEIK (SEQ YYCAR YWPMDSWGQGTLVTVSS ID NO: 84) (SEQ ID NO: 83) C01_Rd4_0.6nM_C16EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSSTYLAWYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLIY DASSRAP GIPDRFSGSGSG WVSAIGgSGGS LPYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQYS SRDNSKNTLYLQMNSLRAEDTAVSWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDS WGQGTLVTVSS NO: 85) (SEQ ID NO:78) C01_Rd4_0.6nM_C09 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSSIFLA WYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLIY DASSRAPGIPDRFSGSGSG WVSAIGgSGGSLPYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQYSSRDNSKNTLYLQMNSLRAEDTAV AWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDSWGQGTLVTVSS NO: 86) (SEQ ID NO: 78) C01_Rd4_6nM_C09EIVLTQSPGTLSLSPGERATLSC A EVQLLESGGGLVQPGGSLRLSCA CSQSVSSTYLAWYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLIY DASSRAP GIPDRFSGSGSGWVSATVgSGGSIGYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQYSSRDNSKNTLYLQMNSLRAEDTAV AWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDSWGQGTLVTVSS NO: 88) (SEQ ID NO: 87) C01_Rd4_0.6nM_C03EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASCDVSSTYLAWYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLIY DASSRAP GIPDRFSGSGSG WVSAIGgSGGS LPYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQY SRDNSKNTLYLQMNSLRAEDTAVMRSPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDS WGQGTLVTVSS NO: 89) (SEQ ID NO:78) C01_Rd4_0.6nM_C06 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASEAVPSTYLA WYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLIY DASSRAPGIPDRFSGSGSG WVSAI GgSGGS LPYADSVKGTISR TDFTLTISRLEPEDFAVYYC QQYSDNSKNTLYLQMNSLRAEDTAVYY AFPLT FGQGTKVEIK (SEQ ID CAR YWPMDS WGQGTLVTVSSNO: 90) (SEQ ID NO: 78) C01_Rd4_6nM_C04 EIVLTQSPGTLSLSPGERATLSC CEVQLLESGGGLVQPGGSLRLSCA SSQSVSSTYLA WYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLELLIY DASSRAP GIPDRFSGSGSG WVSAI GgSGGS LPYADSVKGRFTITDFTLTISRLEPEDFAVYYC QQYS SRDNSKNTLYLQMNSLRAEDTAV AFPLT FGQGTKVEIK (SEQID YYCAR YWPMDS WGQGTLVTVSS NO: 91) (SEQ ID NO: 78) COMBO_Rd4_0.6nM_C22EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASVRVSSTYLAWYQQKPGQAPR ASGFTFS SYAMNWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSG WVSAISdSGGS RWYADSVKGRFT TDFTLTISRLEPEDFAVYYC QQY ISRDNSKNTLYLQMNSLRAEDTAVMKWPLT FGQGTKVEIK (SEQ ID YYCTR YWPMDI WGQGTLVTVSS NO: 93) (SEQ ID NO:92) COMBO_Rd4_6nM_C21 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSAAYLA WYQQKPGQAP ASGFTFS SYPMSWVRQAPGKGLE RLLMY DASIRATGIPDRFSGSGS WVSAI GgSGGS LPYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QQSRDNSKNTLYLQMNSLRAEDTAV YMCWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDSWGQGTLVTVSS NO: 94) (SEQ ID NO: 78) COMBO_Rd4_6nM_C10EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYWG WYQQKPGQAPASGFTFS SYPMSWVRQAPGKGLE RLLMY DASIRAT GIPDRFSGSGS WVSAI GgSGGSIHYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QQ SRDNSKNTLYLQMNSLRAEDTAV YQCWPLTFGQGTKVEIK (SEQ ID YYCAR YWPMDS WGQGTLVTVSS NO: 96) (SEQ ID NO: 95)COMBO_Rd4_0.6nM_C04 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSSTYLA WYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLMY DASIRATGIPDRFSGSGSG WVSAH IgSGGS TYYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQYSRDNSKNTLYLQMNSLRAEDTAV QSWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDSWGQGTLVTVSS NO: 98) (SEQ ID NO: 97) COMBO_Rd4_6nM_C25EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSSpYLAWYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSG WVSAIGgSGGS TYYADSVKGRFTI TDFTLTISRLEPEDFAVYYC QQY SRDNSKNTLYLQMNSLRAEDTAVQSWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDP WGQGTLVTVSS NO: 100) (SEQ ID NO:99) COMBO_Rd4_0.6nM_C21 EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYLA WYQQKPGQAP ASGFTFS SYPMSWVRQAPGKGLERLLMY DASIRAT GIPDRFSGSGS WVSAI GgSGGS LPYADSVKGRFTIGTDFTLTISRLEPEDFAVYYC QQ SRDNSKNTLYLQMNSLRAEDTAV YQSWPLT FGQGTKVEIK (SEQID YYCAR YWPMDS WGQGTLVTVSS NO: 38) (SEQ ID NO: 78) COMBO_Rd4_6nM_C11EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSPIYLAWYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSG WVSAIGGSGGS LGYADSVKGRFT TDFTLTISRLEPEDFAVYYC QQY ISRDNSKNTLYLQMNSLRAEDTAVKAWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDS WGQGTLVTVSS NO: 102) (SEQ ID NO:101) COMBO_Rd4_0.6nM_C20 EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA ASQSVSYLYLA WYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLELLMY DASIRAT GIPDRFSGSGSG WVSAI GGSGGS LPYADSVKGRFT TDFTLTISRLEPEDFAVYYCQQY ISRDNSKNTLYLQMNSLRAEDTAV MEWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDSWGQGTLVTVSS NO: 103) (SEQ ID NO: 78) COMBO_Rd4_6nM_C09EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSAQYLA WYQQKPGQAPASGFTFS SYPMSWVRQAPGKGLE RLLMY DASIRAT GIPDRFSGSGS WVSAI FASGGSTYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QQ SRDNSKNTLYLQMNSLRAEDTAV YQAWPLTFGQGTKVEIK (SEQ ID YYCAR YWPMDS WGQGTLVTVSS NO: 105) (SEQ ID NO: 104)COMBO_Rd4_6nM_C08 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSSSYLA WYQQKPGQAP ASGFTFS SYPMSWVRQAPGKGLE RLLMY DASIRATGIPDRFSGSGS WVSAI GGSGTW TYYADSVKGRFT GTDFTLTISRLEPEDFAVYYC QQISRDNSKNTLYLQMNSLRAEDTAV YQKWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDSWGQGTLVTVSS NO: 107) (SEQ ID NO: 106) COMBO_Rd4_0.6nM_C19EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSAVYLA WYQQKPGQAPASGFTFS SYPMSWVRQAPGKGLE RLLMY DASIRAT GIPDRFSGSGS WVSAI GGSGGSLPYADSVKGRFT GTDFTLTISRLEPEDFAVYYC QQ ISRDNSKNTLYLQMNSLRAEDTAV YRAWPLTFGQGTKVEIK (SEQ ID YYCAR YWPMDS WGQGTLVTVSS NO: 108) (SEQ ID NO: 78)COMBO_Rd4_0.6nM_C02 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASIAVSSTYLA WYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLMY DASIRATGIPDRFSGSGSG WVSAI GGSGGS LPYADSVKGRFT TDFTLTISRLEPEDFAVYYC QQYISRDNSKNTLYLQMNSLRAEDTAV MVWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDSWGQGTLVTVSS NO: 109) (SEQ ID NO: 78) COMBO_Rd4_0.6nM_C23EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA PRQSVSSSYLA WYQQKPGQAPASGFTFS SYPMSWVRQAPGKGLE RLLMY DASIRAT GIPDRFSGSGS WVSAL FGSGGSTYYADSVKGRFT GTDFTLTISRLEPEDFAVYYC QQ ISRDNSKNTLYLQMNSLRAEDTAV YQDWPLTFGQGTKVEIK (SEQ ID YYCAR YWPMDS WGQGTLVTVSS NO: 111) (SEQ ID NO: 110)COMBO_Rd4_0.6nM_C29 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSSSYLA WYQQKPGQAP ASGFTFS SYPMSWVRQAPGKGLE RLLMY DASIRATGIPDRFSGSGS WVSAI GGSGGS LPYADSVKGRFT GTDFTLTISRLEPEDFAVYYC QQISRDNSKNTLYLQMNSLRAEDTAV YQSWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDIWGQGTLVTVSS NO: 38) (SEQ ID NO: 112) COMBO_Rd4_0.6nM_C09EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSSTYLAWYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSG WVSAIGGSGGS LPYADSVKGRFT TDFTLTISRLEPEDFAVYYC QQY ISRDNSKNTLYLQMNSLRAEDTAVQEWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDI WGQGTLVTVSS NO: 113) (SEQ ID NO:112) COMBO_Rd4_6nM_C12 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSASYLA WYQQKPGQAP ASGFTFS SYPMSWVRQAPGKGLE RLLMY DASIRATGIPDRFSGSGS WVSAA LGSGGS TYYADSVKGRF GTDFTLTISRLEPEDFAVYYC QQTISRDNSKNTLYLQMNSLRAEDTA YMSWPLT FGQGTKVEIK (SEQ ID VYYCAR YWPMDSWGQGTLVTVS NO: 115) S (SEQ ID NO: 114) COMBO_Rd4_0.6nM_C30EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSYMYLA WYQQKPGQAPASGFTFS SYPMSWVRQAPGKGLE RLLIY DASIRAT GIPDRFSGSGSG WVSAI GGSGGSTYYADSVKGRFT TDFTLTISRLEPEDFAVYYC QQY ISRDNSKNTLYLQMNSLRAEDTAV KSWPLTFGQGTKVEIK (SEQ ID YYCAR YWPMDS WGQGTLVTVSS NO: 116) (SEQ ID NO: 76)COMBO_Rd4_0.6nM_C14 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSALYLA WYQQKPGQAP ASGFTFS SYPMSWVRQAPGKGLE RLLMY DASIRATGIPDRFSGSGS WVSAI GGSGGS LPYADSVKGRFT GTDFTLTISRLEPEDFAVYYC QQISRDNSKNTLYLQMNSLRAEDTAV YYGWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDIWGQGTLVTVSS NO: 117) (SEQ ID NO: 112) COMBO_Rd4_6nM_C07EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQPISSSYLAWYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSG WVSAIGGSGGS LPYADSVKGRFT TDFTLTISRLEPEDFAVYYC QQY ISRDNSKNTLYLQMNSLRAEDTAVQGWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMAD WGQGTLVTVSS NO: 119) (SEQ ID NO:118) COMBO_Rd4_6nM_C02 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSSSYLA WYQQKPGQAP ASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRATGIPDRFSGSGS WVSAI SDSGGF VYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QQSRDNSKNTLYLQMNSLRAEDTAV YEFWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDSWGQGTLVTVSS NO: 121) (SEQ ID NO: 120) COMBO_Rd4_0.6nM_C05EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSSTYLAWYQQKPGQAPR ASGFTFS SYAMNWVRQAPGKGLE LLMY DASIRAT GIPDRFSGSGSG WVSAIGGSGGS TYYADSVKGRFT TDFTLTISRLEPEDFAVYYC QQY ISRDNSKNTLYLQMNSLRAEDTAVMSWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMSL WGQGTLVTVSS NO: 123) (SEQ ID NO:122) COMBO_Rd4_0.6nM_C17 EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA ASQGISSTYLA WYQQKPGQAPR ASGFTFS SYPMSWVRQAPGKGLELLMY DASIRAT GIPDRFSGSGSG WVSAI GGSGGS LPYADSVKGRFT TDFTLTISRLEPEDFAVYYCQQY ISRDNSKNTLYLQMNSLRAEDTAV AYWPLT FGQGTKVEIK (SEQ ID YYCAR YWPMDIWGQGTLVTVSS NO: 124) (SEQ ID NO: 112) COMBO_Rd4_6nM_C22EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCA ASQSVSSSYLA WYQQKPGQAPASGFTFS SYAMNWVRQAPGKGLE RLLMY DASIRAT GIPDRFSGSGS WVSAC LDSGGSTYYADSVKGRFT GTDFTLTISRLEPEDFAVYYC QQ ISRDNSKNTLYLQMNSLRAEDTAV YQGWPLTFGQGTKVEIK (SEQ ID YYCAR YWPMDS WGQGTLVTVSS NO: 126) (SEQ ID NO: 125)COMBO_Rd4_0.6nM_C11 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSVRYLA WYQQKPGQAP ASGFTFS SYPMSWVRQAPGKGLE RLLMY DASIRATGIPDRFSGSGS WVSAA LGSGGS TYYADSVKGRF GTDFTLTISRLEPEDFAVYYC QQTISRDNSKNTLYLQMNSLRAEDTA YGSWPIT FGQGTKVEIK (SEQ ID VYYCAR YWPMSLWGQGTLVTVS NO: 128) S (SEQ ID NO: 127) Consensus EIVLTQSPGTLSLSPGERATLSCEVQLLESGGGLVQPGGSLRLSCA X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂WYASGFTFX₁SYX₂MX₃WVRQAPGKG QQKPGQAPRLLMYX₁₃ASX₁₄RAX₁₅LEWVSAX₄X₅X₆X₇GX₈X₉X₁₀X₁₁YAD GIPDRFSGSGSGTDFTLTISRLEX₁₂X₁₃KGRFTISRDNSKNTLYLQMN PEDFAVYYCX₁₆X₁₇YX₁₈X₁₉PPSFSLRAEDTAVYYCARVSPIX₁₄X₁₅X₁₆ TFGQGTKVEIK, wherein X₁ is R,MDYWGQGTLVTVSS, wherein X₁ G, W, A, or C; X₂ is A, P, G, L, C, is G orS, X₂ is A or P; X₃ is T, N, or or S; X₃ is S, G, or R; X₄ is Q, C, S;X₄ is I, V, T, H, L, A, or C; X₅ is S, E, V, or I; X₅ is S, P, G, A, R,or D; D, G, T, I, L, F, M, or V; X₆ is G, Y, X₆ is V, G, I, or L; X₇ isS, E, D, P, L, H, D, A, S, or M; X₇ is S, Q, T, A, or G; X₈ is S, P, F,A, M, E, V, N, F, or W; X₈ is G or T; X₉ is N, S, P, D, or Y; X₉ is I,T, V, E, S, A, M, Q, Y, W, or F; X₁₀ is S, T, I, L, T, A, R, Y, H, R, orF; X₁₀ is Y or F; X₁₁ is V, K, G, or C; X₁₁ is F, Y, P, W, H, L, W, orP; X₁₂ is A, S, or G, X₁₃ is or G; X₁₂ is V, R, or L; X₁₃ is G or T; Gor D; X₁₄ is S or I; X₁₅ is T or P; X₁₄ is A or Y; X₁₅ is A or S; andX₁₆ is X₁₆ is Q or K; X₁₇ is H or Y; X₁₈ is G, Q, L, P, or E (SEQ ID NO:313); G, N, or P; and X₁₉ is S, W, or Y or (SEQ ID NO: 315); orEVQLLESGGGLVQPGGSLRLSCA EIVLTQSPGTLSLSPGERATLSC ASGFTFX₁SYX₂MX₃WVRQAPGKGX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂WY LEWVSAX₄X₅X₆X₇GX₈X₉X₁₀X₁₁YADQQKPGQAPRLLMYX₁₃ASX₁₄RAX₁₅ X₁₂X₁₃KGRFTISRDNSKNTLYLQMNGIPDRFSGSGSGTDFTLTISRLE SLRAEDTAVYYCARYWPMX₁₄X₁₅PEDFAVYYCQQYX₁₆X₁₇X₁₈PX₁₉F WGQGTLVTVSS, wherein X₁ is G GQGTKVEIK,wherein X₁ is R, G, or S, X₂ is A or P; X₃ is T, N, or S; W, A, or C; X₂is A, P, G, L, C, or X₄ is I, V, T, H, L, A, or C; X₅ is S, D, S; X₃ isS, G, or R; X₄ is Q, C, E, G, T, I, L, F, M, or V; X₆ is G, Y, L, V, orI; X₅ is S, L, P, G, A, R, or D; H, D, A, S, or M; X₇ is S, Q, T, A, F,X₆ is V, G, or I; X₇ is S, E, D, or P; or W; X₈ is G or T; X₉ is N, S,P, Y, X₈ is S, P, F, A, M, E, V, N, D, or W, or F; X₁₀ is S, T, I, L, T,A, R, V, Y; X₉ is I, T, V, E, S, A, M, Q, Y, H, K, G, or C; X₁₁ is F, Y,P, W, H, or or R; X₁₀ is Y or F; X₁₁ is L, W, or G; X₁₂ is V, R, or L;X₁₃ is G or T; P; X₁₂ is A, S, or G, X₁₃ is G or D; X₁₄ is D, S, T, orA; and X₁₅ is I, S, L, X₁₄ is S or I; X₁₅ is T or P; X₁₆ is G, P, or D(SEQ ID NO: 314) Q, E, L, F, A, S, M, R, K, or Y; X₁₇ is S, R, T, G, R,V, D, A, H, E, K, C, F, or Y; X₁₈ is W, S, or F; and X₁₉ is L or I (SEQID NO: 316) P4G4 EIVLTQSPGTLSLSPGERATLSC R EVQLLESGGGLVQPGGSLRLSCAASQSVSSSYLA WYQQKPGQAP ASGFTFS SYAMSWVRQAPGKGLE RLLIY GASSRAYGIPDRFSGSGS WVSAISASGGSTYYADSVKGRFTI GTDFTLTISRLEPEDFAVYYC QHSRDNSKNTLYLQMNSLRAEDTAV YGSPPLFT FGQGTKVEIK (SEQ YYCAR LSWSGAFDNWGQGTLVT ID NO: 80) VSS (SEQ ID NO: 363) P1A11 EIVLTQSPGTLSLSPGERATLSC REVQLLESGGGLVQPGGSLRLSCA ASQNVSSSYLA WYQQKPGQAP ASGFTFR SYAMSWVRQAPGKGLERLLIY GASYRAT GIPDRFSGSGS WVSAISGSGGSTFYADSVKGRFTI GTDFTLTISRLEPEDFAVYYCQH SRDNSKNTLYLQMNSLRAEDTAV YGSPPSFT FGQGTKVEIK (SEQ YYCAT VGTSGAFGIWGQGTLVTV ID NO: 364) SS (SEQ ID NO: 365)In Table 1, the underlined sequences are CDR sequences according toKabat and in bold according to Chothia, except for the following heavychain CDR2 sequences, in which the Chothia CDR sequences are underlinedand the Kabat CDR sequences are in bold: P5A2_VHVL, A02_Rd4_0.6 nM_C06,A02_Rd4_0.6 nM_C09 A02_Rd4_6 nM_C16, A02_Rd4_6 nM_C03, A02_Rd4_6 nM_C01,A02_Rd4_6 nM_C26 A02_Rd4_6 nM_C25, A02_Rd4_6 nM_C22, A02_Rd4_6 nM_C19,A02_Rd4_0.6 nM_C03 A02_Rd4_6 nM_C07, A02_Rd4_6 nM_C23, A02_Rd4_0.6nM_C18, A02_Rd4_6 nM_C10 A02_Rd4_6 nM_C05, A02_Rd4_0.6 nM_C10, A02_Rd4_6nM_C04, A02 Rd4_0.6 nM C26A02_Rd4_0.6 nM_C13, A02_Rd4_0.6 nM_C01, A02_Rd4_6 nM_C08, P5C1_VHVL,C01_Rd4_6 nM_C24, C01_Rd4_6 nM_C26, C01_Rd4_6 nM_C10, C01_Rd4_0.6 nM_C27C01_Rd4_6 nM_C20, C01_Rd4_6 nM_C12, C01_Rd4_0.6 nM_C16, C01_Rd4_0.6nM_C09C01_Rd4_6 nM_C09, C01_Rd4_0.6 nM_C03, C01_Rd4_0.6 nM_C06, C01_Rd4_6nM_C04COMBO_Rd4_0.6 nM_C22, COMBO_Rd4_6 nM_C21, COMBO_Rd4_6 nM_C10,COMBO_Rd4_0.6 nM_C04, COMBO_Rd4_6 nM_C25, COMBO_Rd4_0.6 nM_C21,COMBO_Rd4_6 nM_C11 COMBO_Rd4_0.6 nM_C20, COMBO_Rd4_6 nM_C09, COMBO_Rd4_6nM_C08, COMBO_Rd4_0.6 nM_C19, COMBO_Rd4_0.6 nM_C02, COMBO_Rd4_0.6nM_C23, COMBO_Rd4_0.6 nM_C29, COMBO_Rd4_0.6 nM_C09, COMBO_Rd4_6 nM_C12,COMBO_Rd4_0.6 nM_C30, COMBO_Rd4_0.6 nM_C14, COMBO_Rd4_6 nM_C07,COMBO_Rd4_6 nM_C02, COMBO_Rd4_0.6 nM_C05, COMBO_Rd4_0.6 nM_C17,COMBO_Rd4_6 nM_C22, and COMBO_Rd4_0.6 nM_C11.

The invention also provides CDR portions of antibodies to BCMA(including Chothia, Kabat CDRs, and CDR contact regions). Determinationof CDR regions is well within the skill of the art. It is understoodthat in some embodiments, CDRs can be a combination of the Kabat andChothia CDR (also termed “combined CRs” or “extended CDRs”). In someembodiments, the CDRs are the Kabat CDRs. In other embodiments, the CDRsare the Chothia CDRs. In other words, in embodiments with more than oneCDR, the CDRs may be any of Kabat, Chothia, combination CDRs, orcombinations thereof. Table 2 provides examples of CDR sequencesprovided herein.

TABLE 2 Heavy Chain mAb CDRH1 CDRH2 CDRH3 P6E01 SYAMT (SEQ ID NO:AISGSGGNTFYADSVKG VSPIASGMDY For the 129) (Kabat); (SEQ ID NO: 132)(Kabat) (SEQ ID NO: 134) following GFTFGSY (SEQ ID SGSGGN (SEQ ID NO:133) mAbs: NO: 130) (Chothia); (Chothia) P6E01/P6E01; GFTFGSYAMT (SEQL1.LGF/ ID NO: 131) L3.KW/P6EO1; (extended) L1.LGF/L3.NY/ P6E01;L1.GDF/L3.NY/ P6E01; L3.KW/P6E01; L3.PY/P6E01; L3.NY/P6E01; L3.PY/L1.PS/P6E01; L3.PY/L1.AH/ P6E01; L3.PY/L1.FF/ P6E01; L3.PY/L1.PH/ P6E01;L3.PY/L3.KY/ P6E01; L3.PY/L3.KF/ P6E01; and L3.PY/P6E01. H3.AQ SYAMT(SEQ ID NO: AISGSGGNTFYADSVKG VSPIAAQMDY For the 129) (Kabat); (SEQ IDNO: 132) (Kabat) (SEQ ID NO: 135) following GFTFGSY (SEQ ID SGSGGN (SEQID NO: 133) mAbs: NO: 130) (Chothia); (Chothia) P6E01/H3.AQ; GFTFGSYAMT(SEQ L1.LGF/L3.KW/ ID NO: 131) H3.AQ; (extended) L1.LGF/L3.PY/ H3.AQ;L1.LGF/L3.NY/ H3.AQ; L1.GDF/L3.KW/ H3.AQ; L1.GDF/L3.PY/ H3.AQ;L1.GDF/L3.NY/ H3.AQ; L3.PY/H3.AQ; L3.PY/L1.PS/ H3.AQ; L3.PY/L1.AH/H3.AQ; L3.PY/L1.FF/ H3.AQ; L3.PY/L1.PH/ H3.AQ; and L3.PY/L3.KF/ H3.AQ.H3.AL SYAMT (SEQ ID NO: AISGSGGNTFYADSVKG VSPIAALMDY For the 129)(Kabat); (SEQ ID NO: 132) (Kabat) (SEQ ID NO: 136) following GFTFGSY(SEQ ID SGSGGN (SEQ ID NO: 133) mAbs: NO: 130) (Chothia); (Chothia)L1.LGF/L3.KW/ GFTFGSYAMT (SEQ H3.AL; ID NO: 131) L1.LGF/L3.NY/(extended) H3.AL; and L1.GDF/L3.NY/ H3.AL. H3.AP SYAMT (SEQ ID NO:AISGSGGNTFYADSVKG VSPIAAPMDY For the 129) (Kabat); (SEQ ID NO: 132)(Kabat) (SEQ ID NO: 137) following GFTFGSY (SEQ ID SGSGGN (SEQ ID NO:133) mAbs: NO: 130) (Chothia); (Chothia) L1.LGF/L3.KW/ GFTFGSYAMT (SEQH3.AP; ID NO: 131) L1.LGF/L3.PY/ (extended) H3.AP; L1.LGF/L3NY/ H3.AP;L1.GDF/L3.KW/ H3.AP; and L1.GDF/L3NY/ H3.AP. H2.QR SYAMT (SEQ ID NO:AISGSGGNTFYADQRKG VSPIASGMDY For the 129) (Kabat); (SEQ ID NO: 138)(Kabat) (SEQ ID NO: 134) following GFTFGSY (SEQ ID SGSGGN (SEQ ID NO:133) mAbs: NO: 130) (Chothia); (Chothia) L3.PY/H2.QR; GFTFGSYAMT (SEQL3.PY/L1.PS/ ID NO: 131) H2.QR; (extended) L3.PY/L1.AH/ H2.QR;L3.PY/L1.FF/ H2.QR; L3.PY/L1.PH/ H2.QR; and L3.PY/L3.KY/ H2.QR. H2.DYSYAMT (SEQ ID NO: AIDYSGGNTFYADSVKG VSPIASGMDY For the 129) (Kabat);(SEQ ID NO: 139) (Kabat) (SEQ ID NO: 134) following GFTFGSY (SEQ IDDYSGGN (SEQ ID NO: 140) mAbs: NO: 130) (Chothia); (Chothia) L3.PY/H2.DY;GFTFGSYAMT (SEQ L3.PY/L1.PS/ ID NO: 131) H2.DY; (extended) L3.PY/L1.AH/H2.DY; L3.PY/L1.FF/ H2.DY; L3.PY/L3.KY/ H2.DY; and L3.PY/L3.KF/ H2.DY.H2.YQ SYAMT (SEQ ID NO: AISYQGGNTFYADSVKG VSPIASGMDY For the 129)(Kabat); (SEQ ID NO: 141) (Kabat) (SEQ ID NO: 134) following GFTFGSY(SEQ ID SYQGGN (SEQ ID NO: 142) mAbs: NO: 130) (Chothia); (Chothia)L3.PY/H2.YQ; GFTFGSYAMT (SEQ L3.PY/L1.PS/ ID NO: 131) H2.YQ; (extended)L3.PY/L1.AH/ H2.YQ; L3.PY/L1.FF/ H2.YQ; L3.PY/L3.KY/ H2.YQ; andL3.PY/L3.KF/ H2.YQ. H2.LT SYAMT (SEQ ID NO: AISLTGGNTFYADSVKG VSPIASGMDYFor the 129) (Kabat); (SEQ ID NO: 143) (Kabat) (SEQ ID NO: 134)following GFTFGSY (SEQ ID SLTGGN (SEQ ID NO: 144) mAbs: NO: 130)(Chothia); (Chothia) L3.PY/H2.LT; GFTFGSYAMT (SEQ L3.PY/L1.PS/ ID NO:131) H2.LT; (extended) L3.PY/L1.AH/ H2.LT; L3.PY/L1.FF/ H2.LT;L3.PY/L3.KY/ H2.LT; and L3.PY/L3.KF/ H2.LT. H2.HA SYAMT (SEQ ID NO:AISHAGGNTFYADSVKG VSPIASGMDY For the 129) (Kabat); (SEQ ID NO: 145)(Kabat) (SEQ ID NO: 134) following GFTFGSY (SEQ ID SHAGGN (SEQ ID NO:146) mAbs: NO: 130) (Chothia); (Chothia) L3.PY/H2.HA; GFTFGSYAMT (SEQL3.PY/L1.AH/ ID NO: 131) H2.HA; (extended) L3.PY/L1.FF/ H2.HA;L3.PY/L1.PH/ H2.HA; and L3.PY/L3.KY/ H2.HA. H2.QL SYAMT (SEQ ID NO:AISGSGGNTFYADQLKG VSPIASGMDY For the 129) (Kabat); (SEQ ID NO: 147)(Kabat) (SEQ ID NO: 134) following GFTFGSY (SEQ ID SGSGGN (SEQ ID NO:133) mAbs: NO: 130) (Chothia); (Chothia) L3.PY/H2.QL; GFTFGSYAMT (SEQL3.PY/L1.PS/ ID NO: 131) H2.QL; (extended) L3.PY/L1.AH/ H2.QL;L3.PY/L1.FF/ H2.QL; L3.PY/L3.KY/ H2.QL; and L3.PY/L3.KF/ H2.QL. H3.YASYAMT (SEQ ID NO: AISGSGGNTFYADSVKG VSPIYAGMDY For the 129) (Kabat);(SEQ ID NO: 132) (Kabat) (SEQ ID NO: 148) following GFTFGSY (SEQ IDSGSGGN (SEQ ID NO: 133) mAbs: NO: 130) (Chothia); (Chothia) L3.PY/H3.YA;GFTFGSYAMT (SEQ L3.PY/L1.PS/ ID NO: 131) H3.YA; (extended) L3.PY/L1.AH/H3.YA; L3.PY/L1.FF/ H3.YA; L3.PY/L3.KY/ H3.YA; and L3.PY/L3.KF/ H3.YA.H3.AE SYAMT (SEQ ID NO: AISGSGGNTFYADSVKG VSPIAAEMDY For the 129)(Kabat); (SEQ ID NO: 132) (Kabat) (SEQ ID NO: 149) following GFTFGSY(SEQ ID SGSGGN (SEQ ID NO: 133) mAbs: NO: 130) (Chothia); (Chothia)L3.PY/H3.AE; GFTFGSYAMT (SEQ L3.PY/L1.AH/ ID NO: 131) H3.AE; (extended)L3.PY/L1.FF/ H3.AE; L3.PY/L1.PH/ H3.AE; and L3.PY/L3.KF/ H3.AE. H3.TAQSYAMT (SEQ ID NO: AISGSGGNTFYADSVKG VSPIAAQMDY For the 129) (Kabat);(SEQ ID NO: 132) (Kabat) (SEQ ID NO: 135) following GFTFGSY (SEQ IDSGSGGN (SEQ ID NO: 133) mAbs: NO: 130) (Chothia); (Chothia)L3.PY/H3.TAQ; GFTFGSYAMT (SEQ L3.PY/L1.PS/ ID NO: 131) H3.TAQ;(extended) L3.PY/L1.AH/ H3.TAQ; L3.PY/L1.FF/ H3.TAQ; L3.PY/L1.PH/H3.TAQ; and L3.PY/L3.KF/ H3.TAQ. P5A2_VH SYAMN (SEQ ID AISDSGGSTYYADSVKGYWPMDI (SEQ VL and NO: 150) (Kabat); (SEQ ID NO: 153) (Kabat) ID NO:155) A02_Rd4_6nM_C03 GFTFSSY (SEQ ID SDSGGS (SEQ ID NO: 154) NO: 151)(Chothia); (Chothia) GFTFSSYAMN (SEQ ID NO: 152) (extended)COMBO_Rd4_0.6nM_C17; SYPMS (SEQ ID NO: AIGGSGGSLPYADSVKG YWPMDI (SEQCOMBO_Rd4_0.6nM_C14; 156) (Kabat); (SEQ ID NO: 158) (Kabat) ID NO: 155)COMBO_Rd4_0.6nM_C29; GFTFSSY (SEQ ID GGSGGS (SEQ ID NO: 159) and NO:151) (Chothia); (Chothia) COMBO_Rd4_0.6nM_C09 GFTFSSYPMS (SEQ ID NO:157) (extended) C01_Rd4_6nM_C04; SYPMS (SEQ ID NO: AIGGSGGSLPYADSVKGYWPMDS (SEQ C01_Rd4_0.6nM_C03; 156) (Kabat); (SEQ ID NO: 158) (Kabat) IDNO: 161) C01_Rd4_0.6nM_C06; GFTFSSY (SEQ ID GGSGGS (SEQ ID NO: 159)COMBO_Rd4_0.6nM_C02; NO: 151) (Chothia); (Chothia) COMBO_Rd4_6nM_C21;GFTFSSYPMS (SEQ C01_Rd4_6nM_C26; ID NO: 157) COMBO_Rd4_0.6nM_C19;(extended) C01_Rd4_6nM_C24; C01_Rd4_6nM_C20; C01_Rd4_0.6nM_C09;COMBO_Rd4_0.6nM_C21; C01_Rd4_0.6nM_C04_C27; C01_Rd4_0.6nM_C16;C01_Rd4_6nM_C10; COMBO_Rd4_0.6nM_C20 P5C1_VHVL SYPMS (SEQ ID NO:AIGGSGGSTYYADSVKG YWPMDS (SEQ and 156) (Kabat); (SEQ ID NO: 162) (Kabat)ID NO: 161) COMBO_Rd4_0.6nM_C30 GFTFSSY (SEQ ID GGSGGS (SEQ ID NO: 159)NO: 151) (Chothia); (Chothia) GFTFSSYPMS (SEQ ID NO: 157) (extended)A02_Rd4_0.6nM_C06 SYAMN (SEQ ID AISDSGGSAWYADSVKG YWPMSL (SEQ NO: 150)(Kabat); (SEQ ID NO: 163) (Kabat) ID NO: 164) GFTFSSY (SEQ ID SDSGGS(SEQ ID NO: 154) NO: 151) (Chothia); (Chothia) GFTFSSYAMN (SEQ ID NO:152) (extended) A02_Rd4_0.6nM_C09 SYAMN (SEQ ID AISDSGGSAWYADSVKG YWPMSL(SEQ NO: 150) (Kabat); (SEQ ID NO: 163) (Kabat) ID NO: 164) GFTFSSY (SEQID SDSGGS (SEQ ID NO: 154) NO: 151) (Chothia); (Chothia) GFTFSSYAMN (SEQID NO: 152) (extended) A02_Rd4_0.6nM_C16 SYAMN (SEQ ID AISDFGGSTYYADSVKGYWPMDI (SEQ NO: 150) (Kabat); (SEQ ID NO: 165) (Kabat) ID NO: 155)GFTFSSY (SEQ ID SDFGGS (SEQ ID NO: 166) NO: 151) (Chothia); (Chothia)GFTFSSYAMN (SEQ ID NO: 152) (extended) A02_Rd4_6nM_C01 SYAMN (SEQ IDAITASGGSTYYADSVKG YWPMSL (SEQ NO: 150) (Kabat); (SEQ ID NO: 167) (Kabat)ID NO: 164) GFTFSSY (SEQ ID TASGGS (SEQ ID NO: 168) NO: 151) (Chothia);(Chothia) GFTFSSYAMN (SEQ ID NO: 152) (extended) A02_Rd4_6nM_C26 SYAMN(SEQ ID AISDSGGSTYYADSVKG YWPMSL (SEQ NO: 150) (Kabat); (SEQ ID NO: 153)(Kabat) ID NO: 164) GFTFSSY (SEQ ID SDSGGS (SEQ ID NO: 154) NO: 151)(Chothia); (Chothia) GFTFSSYAMN (SEQ ID NO: 152) (extended)A02_Rd4_6nM_C25 SYAMN (SEQ ID AISDSGGSRWYADSVKG YWPMTP (SEQ NO: 150)(Kabat); (SEQ ID NO: 169) (Kabat) ID NO: 170) GFTFSSY (SEQ ID SDSGGS(SEQ ID NO: 154) NO: 151) (Chothia); (Chothia) GFTFSSYAMN (SEQ ID NO:152) (extended) A02_Rd4_6nM_C22 SYAMN (SEQ ID AVLDSGGSTYYADSVKG YWPMTP(SEQ NO: 150) (Kabat); (SEQ ID NO: 171) (Kabat) ID NO: 170) GFTFSSY (SEQID LDSGGS (SEQ ID NO: 172) NO: 151) (Chothia); (Chothia) GFTFSSYAMN (SEQID NO: 152) (extended) A02_Rd4_6nM_C19 SYAMN (SEQ ID AISDSGGSRWYADSVKGYWPMSD (SEQ NO: 150) (Kabat); (SEQ ID NO: 169) (Kabat) ID NO: 173)GFTFSSY (SEQ ID SDSGGS (SEQ ID NO: 154) NO: 151) (Chothia); (Chothia)GFTFSSYAMN (SEQ ID NO: 152) (extended) A02_Rd4_0.6nM_C03 SYAMN (SEQ IDAISDSGGSKWYADSVKG YWPMSL (SEQ NO: 150) (Kabat); (SEQ ID NO: 174) (Kabat)ID NO: 164) GFTFSSY (SEQ ID SDSGGS (SEQ ID NO: 154) NO: 151) (Chothia);(Chothia) GFTFSSYAMN (SEQ ID NO: 152) (extended) A02_Rd4_6nM_C07 SYAMN(SEQ ID AIGGSGGSLPYADSVKG(SEQ YWPMDS (SEQ NO: 150) (Kabat); ID NO: 158)(Kabat) ID NO: 161) GFTFSSY (SEQ ID GGSGGS (SEQ ID NO: 159) NO: 151)(Chothia); (Chothia) GFTFSSYAMN (SEQ ID NO: 152) (extended)A02_Rd4_6nM_C23 SYAMN (SEQ ID AISDSGGSGWYADSVKG YWPMSL (SEQ NO: 150)(Kabat); (SEQ ID NO: 175) ID NO: 164) GFTFSSY (SEQ ID SDSGGS (SEQ ID NO:154) NO: 151) (Chothia); (Chothia) GFTFSSYAMN (SEQ ID NO: 152)(extended) A02_Rd4_0.6nM_C18 SYAMN (SEQ ID AVLDSGGSTYYADSVKG YWPMSL (SEQNO: 150) (Kabat); (SEQ ID NO: 171) (Kabat) ID NO: 164) GFTFSSY (SEQ IDLDSGGS (SEQ ID NO: 172) NO: 151) (Chothia); (Chothia) GFTFSSYAMN (SEQ IDNO: 152) (extended) A02_Rd4_6nM_C10 SYAMN (SEQ ID AISDSGGSCWYADSVKGYWPMTP (SEQ NO: 150) (Kabat); (SEQ ID NO: 176) (Kabat) ID NO: 170)GFTFSSY (SEQ ID SDSGGS (SEQ ID NO: 154) NO: 151) (Chothia); (Chothia)GFTFSSYAMN (SEQ ID NO: 152) (extended) A02_Rd4_6nM_C05 SYAMN (SEQ IDAIFASGGSTYYADSVKG YWPMTP (SEQ NO: 150) (Kabat); (SEQ ID NO: 177) (Kabat)ID NO: 170) GFTFSSY (SEQ ID FASGGS (SEQ ID NO: 178) NO: 151) (Chothia);(Chothia) GFTFSSYAMN (SEQ ID NO: 152) (extended) A02_Rd4_0.6nM_C10 SYAMN(SEQ ID AISGWGGSLPYADSVKG YWPMDS (SEQ NO: 150) (Kabat); (SEQ ID NO: 304)(Kabat) ID NO: 161) GFTFSSY (SEQ ID SGWGGS (SEQ ID NO: 179) NO: 151)(Chothia); (Chothia) GFTFSSYAMN (SEQ ID NO: 152) (extended)A02_Rd4_6nM_C04 SYAMN (SEQ ID AIMSSGGPLYYADSVKG YWPMAL (SEQ NO: 150)(Kabat); (SEQ ID NO: 180) (Kabat) ID NO: 182) GFTFSSY (SEQ ID MSSGGP(SEQ ID NO: 181) NO: 151) (Chothia); (Chothia) GFTFSSYAMN (SEQ ID NO:152) (extended) A02_Rd4_0.6nM_C26 SYAMN (SEQ ID AILMSGGSTYYADSVKG YWPMSL(SEQ NO: 150) (Kabat); (SEQ ID NO: 183) (Kabat) ID NO: 164) GFTFSSY (SEQID LMSGGS (SEQ ID NO: 184) NO: 151) (Chothia); (Chothia) GFTFSSYAMN (SEQID NO: 152) (extended) A02_Rd4_0.6nM_C13 SYAMN (SEQ ID AISDSGGYRYYADSVKGYWPMSL (SEQ NO: 150) (Kabat); (SEQ ID NO: 185) (Kabat) ID NO: 164)GFTFSSY (SEQ ID SDSGGY (SEQ ID NO: 186) NO: 151) (Chothia); (Chothia)GFTFSSYAMN (SEQ ID NO: 152) (extended) A02_Rd4_0.6nM_C01 SYAMN (SEQ IDAILSSGGSTYYADSVKG YWPMDI (SEQ NO: 150) (Kabat); (SEQ ID NO: 187) (Kabat)ID NO: 155) GFTFSSY (SEQ ID LSSGGS (SEQ ID NO: 188) NO: 151) (Chothia);(Chothia) GFTFSSYAMN (SEQ ID NO: 152) (extended) A02_Rd4_6nM_C08 SYAMN(SEQ ID AILDSGGSTYYADSVKG YWPMSP (SEQ NO: 150) (Kabat); (SEQ ID NO: 160)(Kabat) ID NO: 189) GFTFSSY (SEQ ID LDSGGS (SEQ ID NO: 172) NO: 151)(Chothia); (Chothia) GFTFSSYAMN (SEQ ID NO: 152) (extended)C01_Rd4_6nM_C12 SYPMS (SEQ ID NO: AIGGSGGWSYYADSVKG YWPMDS (SEQ 156)(Kabat); (SEQ ID NO: 190) (Kabat) ID NO: 161) GFTFSSY (SEQ ID GGSGGW(SEQ ID NO: NO: 151) (Chothia); 191) (Chothia) GFTFSSYPMS (SEQ ID NO:157) (extended) C01_Rd4_6nM_C09 SYPMS (SEQ ID NO: ATVGSGGSIGYADSVKGYWPMDS (SEQ 156) (Kabat); (SEQ ID NO: 192) (Kabat) ID NO: 161) GFTFSSY(SEQ ID VGSGGS (SEQ ID NO: 193) NO: 151) (Chothia); (Chothia) GFTFSSYPMS(SEQ ID NO: 157) (extended) COMBO_Rd4_0.6nM_C22 SYAMN (SEQ IDAISDSGGSRWYADSVKG YWPMDI (SEQ NO: 150) (Kabat); (SEQ ID NO: 169) (Kabat)ID NO: 155) GFTFSSY (SEQ ID SDSGGS (SEQ ID NO: 154) NO: 151) (Chothia);(Chothia) GFTFSSYAMN (SEQ ID NO: 152) (extended) COMBO_Rd4_0.6nM_C10SYPMS (SEQ ID NO: AIGGSGGSIHYADSVKG YWPMDS (SEQ 156) (Kabat); (SEQ IDNO: 194) (Kabat) ID NO: 161) GFTFSSY (SEQ ID GGSGGS (SEQ ID NO: 159) NO:151) (Chothia); (Chothia) GFTFSSYPMS (SEQ ID NO: 157) (extended)COMBO_Rd4_0.6nM_C04 SYPMS (SEQ ID NO: AHIGSGGSTYYADSVKG YWPMDS (SEQ 156)(Kabat); (SEQ ID NO: 195) (Kabat) ID NO: 161) GFTFSSY (SEQ ID IGSGGS(SEQ ID NO: 196) NO: 151) (Chothia); (Chothia) GFTFSSYPMS (SEQ ID NO:157) (extended) COMBO_Rd4_0.6nM_C25 SYPMS (SEQ ID NO: AIGGSGGSTYYADSVKGYWPMDP (SEQ 156) (Kabat); (SEQ ID NO: 162) (Kabat) ID NO: 197) GFTFSSY(SEQ ID GGSGGS (SEQ ID NO: 159 NO: 151) (Chothia); (Chothia) GFTFSSYPMS(SEQ ID NO: 157) (extended) COMBO_Rd4_6nM_C21 SYPMS (SEQ ID NO:AIGGSGGSLPYADSVKG YWPMDS (SEQ 156) (Kabat); (SEQ ID NO: 158) (Kabat) IDNO: 161) GFTFSSY (SEQ ID GGSGGS (SEQ ID NO: 159) NO: 151) (Chothia);(Chothia) GFTFSSYPMS (SEQ ID NO: 157) (extended) COMBO_Rd4_6nM_C11 SYPMS(SEQ ID NO: AIGGSGGSLGYADSVKG YWPMDS (SEQ 156) (Kabat); (SEQ ID NO: 198)(Kabat) ID NO: 161) GFTFSSY (SEQ ID GGSGGS (SEQ ID NO: 159) NO: 151)(Chothia); (Chothia) GFTFSSYPMS (SEQ ID NO: 157) (extended)COMBO_Rd4_6nM_C09 SYPMS (SEQ ID NO: AIFASGGSTYYADSVKG YWPMDS (SEQ 156)(Kabat); (SEQ ID NO: 177) (Kabat) ID NO: 161) GFTFSSY (SEQ ID FASGGS(SEQ ID NO: 178) NO: 151) (Chothia); (Chothia) GFTFSSYPMS (SEQ ID NO:157) (extended) COMBO_Rd4_6nM_C08 SYPMS (SEQ ID NO: AIGGSGTWTYYADSVKGYWPMDS (SEQ 156) (Kabat); (SEQ ID NO: 199) (Kabat) ID NO: 161) GFTFSSY(SEQ ID GGSGTW (SEQ ID NO: 200) NO: 151) (Chothia); (Chothia) GFTFSSYPMS(SEQ ID NO: 157) (extended) COMBO_Rd4_0.6nM_C23 SYPMS (SEQ ID NO:ALFGSGGSTYYADSVKG YWPMDS (SEQ 156) (Kabat); (SEQ ID NO: 201) (Kabat) IDNO: 161) GFTFSSY (SEQ ID FGSGGS NO: 151) (Chothia); (SEQ ID NO: 202)(Chothia) GFTFSSYPMS (SEQ ID NO: 157) (extended) COMBO_Rd4_0.6nM_C12SYPMS (SEQ ID NO: AALGSGGSTYYADSVKG YWPMDS (SEQ 156) (Kabat); (SEQ IDNO: 203) (Kabat) ID NO: 161) GFTFSSY (SEQ ID LGSGGS (SEQ ID NO: 204) NO:151) (Chothia); (Chothia) GFTFSSYPMS (SEQ ID NO: 157) (extended)COMBO_Rd4_6nM_C07 SYPMS (SEQ ID NO: AIGGSGGSLPYADSVKG YWPMAD (SEQ 156)(Kabat); (SEQ ID NO: 158) (Kabat) ID NO: 205) GFTFSSY (SEQ ID GGSGGS(SEQ ID NO: 159) NO: 151) (Chothia); (Chothia) GFTFSSYPMS (SEQ ID NO:157) (extended) COMBO_Rd4_6nM_C02 SYAMN (SEQ ID AISDSGGFVYYADSVKG YWPMDS(SEQ NO: 150) (Kabat); (SEQ ID NO: 206) (Kabat) ID NO: 161) GFTFSSY (SEQID SDSGGF (SEQ ID NO: 207) NO: 151) (Chothia); (Chothia) GFTFSSYAMN (SEQID NO: 152) (extended) COMBO_Rd4_6nM_C05 SYAMN (SEQ ID AIGGSGGSTYYADSVKGYWPMSL (SEQ NO: 150) (Kabat); (SEQ ID NO: 162) (Kabat) ID NO: 164)GFTFSSY (SEQ ID GGSGGS (SEQ ID NO: 159) NO: 151) (Chothia); (Chothia)GFTFSSYAMN (SEQ ID NO: 152) (extended) COMBO_Rd4_6nM_C22 SYAMN (SEQ IDACLDSGGSTYYADSVKG YWPMDS (SEQ NO: 150) (Kabat); (SEQ ID NO: 208) (Kabat)ID NO: 161) GFTFSSY (SEQ ID LDSGGS (SEQ ID NO: 172) NO: 151) (Chothia);(Chothia) GFTFSSYAMN (SEQ ID NO: 152) (extended) COMBO_Rd4_6nM_C11 SYPMS(SEQ ID NO: AALGSGGSTYYADSVKG YWPMSL (SEQ 156) (Kabat); (SEQ ID NO: 203)(Kabat) ID NO: 164) GFTFSSY (SEQ ID LGSGGS (SEQ ID NO: 204) NO: 151)(Chothia); (Chothia) GFTFSSYPMS (SEQ ID NO: 157) (extended) HeavySYX₁MX₂, wherein AX₁X₂X₃X₄GX₅X₆X₇X₈YADX₉ VSPIX₁X₂X₃MDY, chain X₁ is A orP; and X₂ X₁₀KG, wherein X₁ is I, V, T, wherein X₁ is A or consensus isT, N, or S (Kabat) H, L, A, or C; X₂ is S, D, G, Y; X₂ is A or S; (SEQID NO: 301) T, I, L, F, M, or V; X₃ is G, Y, and X₃ is G, Q, L,GFTFX₁SY, wherein L, H, D, A, S, or M; X₄ is S, P, or E (SEQ ID X₁ is Gor S (Chothia) Q, T, A, F, or W; X₅ is G or NO: 307) (SEQ ID NO: 302) T;X₆ is N, S, P, Y, W, or F; YWPMX₁X₂, GFTFX₁SYX₂MX₃, X₇ is S, T, I, L, A,R, V, K, G, wherein X₁ is D, wherein X₁ is G or S, or C; X₈ is F, Y, P,W, H, or S, T, or A; and X₂ X₂ is A or P; and X₃ G; X₉ is V, R, or L;and X₁₀ is is I, S, L, P, or D is T, N, or S (SEQ ID G or T (Kabat) (SEQID NO: (SEQ ID NO: 308) NO: 303) (extended) 305) X₁X₂X₃X₄X₅X₆, whereinX₁ is S, V, I, D, G, T, L, F, or M; X₂ is G, Y, L, H, D, A, S, or M; X₃is S, G, F, or W; X₄ is G or S; X₅ is G or T; and X₆ is N, S, P, Y, or W(Chothia) (SEQ ID NO: 306) P4G4 SYAMS (SEQ ID NO: SASGGS (SEQ ID NO:368) LSWSGAFDN 366) (Kabat); (Chothia) (SEQ ID NO: 370) GFTFSSY (SEQ IDAISASGGSTYYADSVKG NO: 151) (Chothia); (SEQ ID NO: 369) (Kabat)GFTFSSYAMS (SEQ ID NO: 367) (extended) P1A11 SYAMS (SEQ ID NO: SGSGGS(SEQ ID NO: 359) VGTSGAFGI 366) (Kabat); (Chothia) (SEQ ID NO: 361)GFTFRSY (SEQ ID AISGSGGSTFYADSVKG NO: 371) (SEQ ID NO: 360) (Kabat)GFTFRSYAMS (SEQ ID NO: 372) Light Chain mAb CDRL1 CDRL2 CDRL3 P6E01RASQSVSSSYLA GASSRAT (SEQ ID NO: QHYGSPPSFT For the (SEQ ID NO: 209)210) (SEQ ID NO: 211) following mAbs: P6E01/P6E01; and P6E01/H3.AQ.L1.LGF/ RASQSLGSFYLA GASSRAT (SEQ ID NO: KHYGWPPSFT L3.KW (SEQ ID NO:212) 210) (SEQ ID NO: 213) For the following mAbs: L1.LGF/L3.KW/ P6E01;L1.LGF/L3.KW/ H3.AL; L1.LGF/L3.KW/ H3.AP; and L1.LGF/L3.KW/ H3.AQL1.LGF/ RASQSLGSFYLA GASSRAT (SEQ ID NO: QHYNYPPSFT L3.NY (SEQ ID NO:212) 210) (SEQ ID NO: 214) For the following mAbs: L1.LGF/L3.NY/ P6E01;L1.LGF/L3.NY/ H3.AL; L1.LGF/L3.NY/ H3.AP; and L1.LGF/L3.NY/ H3AQ L1.GDF/RASQSVGDFYLA GASSRAT (SEQ ID NO: QHYNYPPSFT L3.NY (SEQ ID NO: 215) 210)(SEQ ID NO: 214) For the following mAbs: L1.GDF/L3.NY/ P6E01;L1.GDF/L3.NY/ H3.AL; L1.GDF/L3.NY/ H3.AP; and L1.GDF/L3.NY/ H3.AQL1.LGF/ RASQSLGSFYLA GASSRAT (SEQ ID NO: QHYPYPPSFT L3.PY (SEQ ID NO:212) 210) (SEQ ID NO: 216) For the following mAbs: L1.LGF/L3.PY/ H3.AP;and L1.LGF/L3.PY/ H3.AQ L1.GDF/ RASQSVGDFYLA GASSRAT (SEQ ID NO:KHYGWPPSFT L3.KW (SEQ ID NO: 215) 210) (SEQ ID NO: 213) For thefollowing mAbs: L1.GDF/ L3.KW/H3.AL; L1.GDF/ L3.KW/H3.AP; and L1.GDF/L3.KW/H3.AQ L1.GDF/ RASQSVGDFYLA GASSRAT (SEQ ID NO: QHYPYPPSFTL3.PY/H3.AQ (SEQ ID NO: 215) 210) (SEQ ID NO: 216) L3.KW/P6E01RASQSVSSSYLA GASSRAT (SEQ ID NO: KHYGWPPSFT (SEQ ID NO: 209) 210) (SEQID NO: 213) L3.PY RASQSVSSSYLA GASSRAT (SEQ ID NO: QHYPYPPSFT For the(SEQ ID NO: 209) 210) (SEQ ID NO: 216) following mAbs: L3.PY/P6E01;L3.PY/H2.QR; L3.PY/H2.DY; L3.PY/H2.YQ; L3.PY/H2.LT; L3.PY/H2.HA;L3.PY/H2.QL; L3.PY/H3.YA; L3.PY/H3.AE; L3.PY/H3.AQ; L3.PY/H3.TAQL3.NY/P6E01 RASQSVSSSYLA GASSRAT (SEQ ID NO: QHYNYPPSFT (SEQ ID NO: 209)210) (SEQ ID NO: 214) L3.PY/L1.PS RASQSVSSSYPS GASSRAT (SEQ ID NO:QHYPYPPSFT For the (SEQ ID NO: 217) 210) (SEQ ID NO: 216) followingmAbs: L3.PY/L1.PS/ P6E01; L3.PY/L1.PS/ H2.QR; L3.PY/L1.PS/ H2.DY;L3.PY/L1.PS/ H2.YQ; L3.PY/L1.PS/ H2.LT; L3.PY/L1.PS/ H2.HA; L3.PY/L1.PS/H2.QL; L3.PY/L1.PS/ H3.YA; L3.PY/L1.PS/ H3.AE; L3.PY/L1.PS/ H3.AQ;L3.PY/L1.PS/ H3.TAQ L3.PY/L1.AH RASQSVSAHYLA GASSRAT (SEQ ID NO:QHYPYPPSFT For the (SEQ ID NO: 218) 210) (SEQ ID NO: 216) followingmAbs: L3.PY/L1.AH/ P6E01; L3.PY/L1.AH/ H2.QR; L3.PY/L1.AH/ H2.DY;L3.PY/L1.AH/ H2.YQ; L3.PY/L1.AH/ H2.LT; L3.PY/L1.AH/ H2.HA; L3.PY/L1.AH/H2.QL; L3.PY/L1.AH/ H3.YA; L3.PY/L1.AH/ H3.AE; L3.PY/L1.AH/ H3.AQ;L3.PY/L1.AH/ H3.TAQ L3.PY/L1.FF RASQSVSSFFLA GASSRAT (SEQ ID NO:QHYPYPPSFT For the (SEQ ID NO: 219) 210) (SEQ ID NO: 216) followingmAbs: L3.PY/L1.FF/ P6E01; L3.PY/L1.FF/ H2.QR; L3.PY/L1.FF/ H2.DY;L3.PY/L1.FF/ H2.YQ; L3.PY/L1.FF/ H2.LT; L3.PY/L1.FF/ H2.HA; L3.PY/L1.FF/H2.QL; L3.PY/L1.FF/ H3.YA; L3.PY/L1.FF/ H3.AE; L3.PY/L1.FF/ H3.AQ; andL3.PY/L1.FF/ H3.TAQ L3.PY/L1.PH RASQSVSPHYLA GASSRAT (SEQ ID NO:QHYPYPPSFT For the (SEQ ID NO: 219) 210) (SEQ ID NO: 216) followingmAbs: L3.PY/L1.PH/ P6E01; L3.PY/L1.PH/ H2.QR; L3.PY/L1.PH/ H2.HA;L3.PY/L1.PH/ H3.AE; L3.PY/L1.PH/ H3.AQ; and L3.PY/L1.PH/ H3.TAQL3.PY/L3.KY RASQSVSSSYLA GASSRAT (SEQ ID NO: KYYPYPPSFT For the (SEQ IDNO: 209) 210) (SEQ ID NO: 220) following mAbs: L3.PY/L3.KY/ P6E01;L3.PY/L3.KY/ H2.QR; L3.PY/L3.KY/ H2.DY; L3.PY/L3.KY/ H2.YQ; L3.PY/L3.KY/H2.LT; L3.PY/L3.KY/ H2.HA; L3.PY/L3.KY/ H2.QL; L3.PY/L3.KY/ H3.YA; andL3.PY/L3.KY/ H3.TAQ L3.PY/L3.KF RASQSVSSSYLA GASSRAT (SEQ ID NO:KFYPYPPSFT (SEQ For the (SEQ ID NO: 209) 210) ID NO: 220) followingmAbs: L3.PY/L3.KF/ H2.DY; L3.PY/L3.KF/ H2.YQ; L3.PY/L3.KF/ H2.LT;L3.PY/L3.KF/ H2.QL; L3.PY/L3.KF/ H3.YA; L3.PY/L3.KF/ H3.AE; L3.PY/L3.KF/H3.AQ; and L3.PY/L3.KF/ H3.TAQ P5A2_VHVL RASQSVSSSYLA DASIRAT QQYGSWPLT(SEQ (SEQ ID NO: 209) (SEQ ID NO: 221) ID NO: 222) A02_Rd4_0.6nM_C06RASQSVSVIYLA DASIRAT QQYQRWPLT (SEQ ID NO: 223) (SEQ ID NO: 221) (SEQ IDNO: 224) A02_Rd4_0.6nM_C09; RASQSVSSSYLA DASIRAT QQYQSWPLTCOMBO_Rd_0.6nM_C29; (SEQ ID NO: 209) (SEQ ID NO: 221) (SEQ ID NO: 225)and COMBO_Rd4_0.6nM_C21 A02_Rd4_6nM_C16 RASQSVSDIYLA DASIRAT QQYQTWPLT(SEQ (SEQ ID NO: 226) (SEQ ID NO: 221) ID NO: 227) A02_Rd4_6nM_C03RASQSVSNIYLA DASIRAT QQYQGWPLT (SEQ ID NO: 228) (SEQ ID NO: 221) (SEQ IDNO: 229) A02_Rd4_6nM_C01 RASQSVSAYYLA DASIRAT QQYERWPLT (SEQ ID NO: 230)(SEQ ID NO: 221) (SEQ ID NO: 231) A02_Rd4_6nM_C26 RASQSVSSIYLA DASIRATQQYQVWPLT (SEQ ID NO: 232) (SEQ ID NO: 221) (SEQ ID NO: 233)A02_Rd4_6nM_C25 RASQSVSSSYLA DASIRAT QQYLDWPLT (SEQ ID NO: 209) (SEQ IDNO: 221) (SEQ ID NO: 234) A02_Rd4_6nM_C22 RASQSVSSSYLA DASIRAT QQYQVWPLT(SEQ ID NO: 209) (SEQ ID NO: 221) (SEQ ID NO: 233) A02_Rd4_6nM_C19RASQSVSVIYLA DASIRAT QQYLAWPLT (SEQ ID NO: 223) (SEQ ID NO: 221) (SEQ IDNO: 236) A02_Rd4_0.6nM_C03 RASQSVSSSYLA DASIRAT QQYFTWPLT (SEQ ID NO:209) (SEQ ID NO: 221) (SEQ ID NO: 237) A02_Rd4_6nM_C07 RASQSVSPYYLADASIRAT QQYERWPLT (SEQ ID NO: 238) (SEQ ID NO: 221) (SEQ ID NO: 231)A02_Rd4_6nM_C23 RASQSVSVEYLA DASIRAT QQYARWPLT (SEQ ID NO: 239) (SEQ IDNO: 221) (SEQ ID NO: 240) A02_Rd4_0.6nM_C18 RASQSVSEIYLA DASIRATQQYFGWPLT (SEQ ID NO: 241) (SEQ ID NO: 221) (SEQ ID NO: 242)A02_Rd4_6nM_C10 RASQSVEMSYLA DASIRAT QQYAHWPLT (SEQ ID NO: 243) (SEQ IDNO: 221) (SEQ ID NO: 244) A02_Rd4_6nM_C05 RASQSVSSSYLA DASIRAT QQYQRWPLT(SEQ ID NO: 209) (SEQ ID NO: 221) (SEQ ID NO: 224) A02_Rd4_0.6nM_C10RASQSVSAQYLA DASIRAT QQYQRWPLT (SEQ ID NO: 245) (SEQ ID NO: 221) (SEQ IDNO: 224) A02_Rd4_6nM_C04 RASQSVSAIYLA DASIRAT QQYQVWPLT (SEQ ID NO: 235)(SEQ ID NO: 221) (SEQ ID NO: 233) A02_Rd4_0.6nM_C26 GPSQSVSSSYLA DASIRATQQYQSWPLT (SEQ ID NO: 246) (SEQ ID NO: 221) (SEQ ID NO: 225)A02_Rd4_0.6nM_C13 RASQSVSSSYWA DASIRAT QQYESWPLT (SEQ ID NO: 247) (SEQID NO: 221) (SEQ ID NO: 248) A02_Rd4_0.6nM_C01 RGGQSVSSSYLA DASIRATQQYQSWPLT (SEQ ID NO: 249) (SEQ ID NO: 221) (SEQ ID NO: 225)A02_Rd4_6nM_C08 RASQSVSFIYLA DASIRAT QQYGSWPLT (SEQ (SEQ ID NO: 250)(SEQ ID NO: 221) ID NO: 222) P5C1_VHVL RASQSVSSTYLA DASSRAP QQYSTSPLT(SEQ ID NO: 251) (SEQ ID NO: 252) (SEQ ID NO: 253) C01_Rd4_6nM_C24RASQSVSPEYLA DASSRAP QQYSVWPLT (SEQ ID NO: 254) (SEQ ID NO: 252) (SEQ IDNO: 255) C01_Rd4_6nM_C26 RASQSVSAIYLA DASSRAP QQYSAWPLT (SEQ ID NO: 235)(SEQ ID NO: 252) (SEQ ID NO: 256) C01_Rd4_6nM_C10 RASQSVSSVYLA DASSRAPQQYSTWPLT (SEQ ID NO: 257) (SEQ ID NO: 252) (SEQ ID NO: 258)C01_Rd4_0.6nM_C27 RASQSVSSTYLA DASSRAP QQYSRWPLT (SEQ ID NO: 251) (SEQID NO: 252) (SEQ ID NO: 259) C01_Rd4_6nM_C20 RASQSVSPIYLA DASSRAPQQYSAFPLT (SEQ ID NO: 260) (SEQ ID NO: 252) (SEQ ID NO: 261)C01_Rd4_6nM_C12 WLSQSVSSTYLA DASSRAP QQYSEWPLT (SEQ ID NO: 262) (SEQ IDNO: 252) (SEQ ID NO: 263) C01_Rd4_0.6nM_C16 RASQSVSSTYLA DASSRAPQQYSSWPLT (SEQ ID NO: 251) (SEQ ID NO: 252) (SEQ ID NO: 264)C01_Rd4_0.6nM_C09 RASQSVSSIFLA DASSRAP QQYSAWPLT (SEQ ID NO: 265) (SEQID NO: 252) (SEQ ID NO: 256) C01_Rd4_6nM_C09 ACSQSVSSTYLA DASSRAPQQYSAWPLT (SEQ ID NO: 266) (SEQ ID NO: 252) (SEQ ID NO: 256)C01_Rd4_0.6nM_C03 RASCDVSSTYLA DASSRAP QQYMRSPLT (SEQ ID NO: 267) (SEQID NO: 252) (SEQ ID NO: 268) C01_Rd4_0.6nM_C06 RASEAVPSTYLA DASSRAPQQYSAFPLT (SEQ ID NO: 269) (SEQ ID NO: 252) (SEQ ID NO: 261)C01_Rd4_0.6nM_C04 CSSQSVSSTYLA DASSRAP QQYSAFPLT (SEQ ID NO: 270) (SEQID NO: 252) (SEQ ID NO: 261) COMBO_Rd4_0.6nM_C22 RASVRVSSTYLA DASIRATQQYMKWPLT (SEQ ID NO: 271) (SEQ ID NO: 221) (SEQ ID NO: 272)COMBO_Rd4_6nM_C21 RASQSVSAAYLA DASIRAT QQYMCWPLT (SEQ ID NO: 273) (SEQID NO: 221) (SEQ ID NO: 274) COMBO_Rd4_6nM_C10 RASQSVSSSYWG DASIRATQQYQCWPLT (SEQ ID NO: 275) (SEQ ID NO: 221) (SEQ ID NO: 276)COMBO_Rd4_0.6nM_C04 RASQSVSSTYLA DASIRAT QQYQSWPLT (SEQ ID NO: 251) (SEQID NO: 221) (SEQ ID NO: 225) COMBO_Rd4_6nM_C25 RASQSVSSPYLA DASIRATQQYQSWPLT (SEQ ID NO: 277) (SEQ ID NO: 221) (SEQ ID NO: 225)COMBO_Rd4_6nM_C11 RASQSVSPIYLA DASIRAT QQYKAWPLT (SEQ ID NO: 260) (SEQID NO: 221) (SEQ ID NO: 278) COMBO_Rd4_0.6nM_C20 RASQSVSYLYLA DASIRATQQYMEWPLT (SEQ ID NO: 279) (SEQ ID NO: 221) (SEQ ID NO: 280)COMBO_Rd4_6nM_C09 RASQSVSAQYLA DASIRAT QQYQAWPLT (SEQ ID NO: 245) (SEQID NO: 221) (SEQ ID NO: 281) COMBO_Rd4_6nM_C08 RASQSVSSSYLA DASIRATQQYQKWPLT (SEQ ID NO: 209) (SEQ ID NO: 221) (SEQ ID NO: 282)COMBO_Rd4_0.6nM_C19 RASQSVSAVYLA DASIRAT QQYRAWPLT (SEQ ID NO: 283) (SEQID NO: 221) (SEQ ID NO: 284) COMBO_Rd4_0.6nM_C02 RASIAVSSTYLA DASIRATQQYMVWPLT (SEQ ID NO: 285) (SEQ ID NO: 221) (SEQ ID NO: 286)COMBO_Rd4_0.6nM_C23 RPRQSVSSSYLA DASIRAT QQYQDWPLT (SEQ ID NO: 287) (SEQID NO: 221) (SEQ ID NO: 288) COMBO_Rd4_0.6nM_C09 RASQSVSSTYLA DASIRATQQYQEWPLT (SEQ ID NO: 251) (SEQ ID NO: 221) (SEQ ID NO: 289)COMBO_Rd4_6nM_C12 RASQSVSASYLA DASIRAT QQYMSWPLT (SEQ ID NO: 290) (SEQID NO: 221) (SEQ ID NO: 291) COMBO_Rd4_0.6nM_C30 RASQSVSYMYLA DASIRATQQYKSWPLT (SEQ ID NO: 292) (SEQ ID NO: 221) (SEQ ID NO: 293)COMBO_Rd4_0.6nM_C14 RASQSVSAIYLA DASIRAT QQYYGWPLT (SEQ ID NO: 235) (SEQID NO: 221) (SEQ ID NO: 294) COMBO_Rd4_6nM_C07 RASQPISSSYLA DASIRATQQYQGWPLT (SEQ ID NO: 295) (SEQ ID NO: 221) (SEQ ID NO: 229)COMBO_Rd4_6nM_C02 RASQSVSSSYLA DASIRAT QQYEFWPLT (SEQ ID NO: 209) (SEQID NO: 221) (SEQ ID NO: 296) COMBO_Rd4_0.6nM_C05 RASQSVSSTYLA DASIRATQQYMSWPLT (SEQ ID NO: 251) (SEQ ID NO: 221) (SEQ ID NO: 291)COMBO_Rd4_0.6nM_C17 RASQGISSTYLA DASIRAT QQYAYWPLT (SEQ ID NO: 297) (SEQID NO: 221) (SEQ ID NO: 298) COMBO_Rd4_6nM_C22 RASQSVSSSYLA DASIRATQQYQGWPLT (SEQ ID NO: 209) (SEQ ID NO: 221) (SEQ ID NO: 229)COMBO_Rd4_0.6nM_C11 RASQSVSVRYLA DASIRAT QQYGSWPIT (SEQ ID NO: 299) (SEQID NO: 221) (SEQ ID NO: 300) Light X₁X₂X₃X₄X₅X₆X₇X₈X₉ X₁ASX₂RAX₃,wherein X₁ X₁X₂YX₃X₄PPSFT, chain X₁₀X₁₁X₁₂, wherein X₁ is G or D; X₂ isS or I; and wherein X₁ is Q or K; consensus is R, G, W, A, or C; X₂ X₃is T or P (SEQ ID NO: X₂ is H or Y; X₃ is G, is A, P, G, L, C, or S;310) N, or P; and X₄ is S, X₃ is S, G, or R; X₄ is W, or Y (SEQ ID Q, C,E, V, or I; X₅ is NO: 311) S, P, G, A, R, or D; X₆ QQYX₁X₂X₃PX₄T, is V,G, I, or L; X₇ is wherein X₁ is G, Q, S, E, D, P, or G; X₈ is E, L, F,A, S, M, K, S, P, F, A, M, E, V, N, R, or Y; X₂ is S, R, D, or Y; X₉ isI, T, V, T, G, V, F, Y, D, A, E, F H, V, E, K, or C; X₃ S, A, M, Q, Y,H, or R; is W, F, or S; and X₄ X₁₀ is Y or F; X₁₁ is L, is L or I (SEQID W, or P; and X₁₂ is A, NO: 312) S, or G (SEQ ID NO: 309) P4G4RASQSVSSSYLA GASSRAY (SEQ ID NO: QHYGSPPLFT (SEQ ID NO: 209) 362) (SEQID NO: 499) P1A11 RASQNVSSSYLA GASYRAT (SEQ ID NO: QHYGSPPSFT (SEQ IDNO: 500) 501) (SEQ ID NO: 211)

In some embodiments, the present invention provides an antibody thatbinds to BCMA and competes with the antibody as described herein,including P6E01/P6E01, P6E01/H3.AQ, L1.LGF/L3.KW/P6E01;L1.LGF/L3.NY/P6E01, L1.GDF/L3.NY/P6E01, L1.LGF/L3.KW/H3.AL,L1.LGF/L3.KW/H3.AP, L1.LGF/L3.KW/H3.AQ, L1.LGF/L3.PY/H3.AP,L1.LGF/L3.PY/H3.AQ, L1.LGF/L3.NY/H3.AL, L1.LGF/L3.NY/H3.AP,L1.LGF/L3.NY/H3.AQ, L1.GDF/L3.KW/H3.AL, L1.GDF/L3.KW/H3.AP,L1.GDF/L3.KW/H3.AQ, L1.GDF/L3.PY/H3.AQ, L1.GDF/L3.NY/H3.AL,L1.GDF/L3.NY/H3.AP, L1.GDF/L3.NY/H3.AQ, L3.KW/P6E01, L3.PY/P6E01,L3.NY/P6E01,

L3.PY/L1.PS/P6E01, L3.PY/L1.AH/P6E01, L3.PY/L1.FF/P6E01,L3.PY/L1.PH/P6E01, L3.PY/L3.KY/P6E01, L3.PY/L3.KF/P6E01, L3.PY/H2.QR,L3.PY/H2.DY, L3.PY/H2.YQ, L3.PY/H2.LT, L3.PY/H2.HA, L3.PY/H2.QL,L3.PY/H3.YA, L3.PY/H3.AE, L3.PY/H3.AQ, L3.PY/H3.TAQ, L3.PY/P6E01,L3.PY/L1.PS/H2.QR, L3.PY/L1.PS/H2.DY, L3.PY/L1.PS/H2.YQ,L3.PY/L1.PS/H2.LT, L3.PY/L1.PS/H2.HA, L3.PY/L1.PS/H2.QL,L3.PY/L1.PS/H3.YA, L3.PY/L1.PS/H3.AE, L3.PY/L1.PS/H3.AQ,L3.PY/L1.PS/H3.TAQ, L3.PY/L1.AH/H2.QR, L3.PY/L1.AH/H2.DY,L3.PY/L1.AH/H2.YQ, L3.PY/L1.AH/H2.LT, L3.PY/L1.AH/H2.HA,L3.PY/L1.AH/H2.QL, L3.PY/L1.AH/H3.YA, L3.PY/L1.AH/H3.AE,L3.PY/L1.AH/H3.AQ, L3.PY/L1.AH/H3.TAQ, L3.PY/L1.FF/H2.QR,L3.PY/L1.FF/H2.DY, L3.PY/L1.FF/H2.YQ, L3.PY/L1.FF/H2.LT,L3.PY/L1.FF/H2.HA, L3.PY/L1.FF/H2.QL, L3.PY/L1.FF/H3.YA,L3.PY/L1.FF/H3.AE, L3.PY/L1.FF/H3.AQ, L3.PY/L1.FF/H3.TAQ,L3.PY/L1.PH/H2.QR, L3.PY/L1.PH/H2.HA, L3.PY/L1.PH/H3.AE,L3.PY/L1.PH/H3.AQ, L3.PY/L1.PH/H3.TAQ, L3.PY/L3.KY/H2.QR,L3.PY/L3.KY/H2.DY, L3.PY/L3.KY/H2.YQ L3.PY/L3.KY/H2.LT,L3.PY/L3.KY/H2.HA, L3.PY/L3.KY/H2.QL, L3.PY/L3.KY/H3.YAL3.PY/L3.KY/H3.TAQ, L3.PY/L3.KF/H2.DY, L3.PY/L3.KF/H2.YQ,L3.PY/L3.KF/H2.LT L3.PY/L3.KF/H2.QL, L3.PY/L3.KF/H3.YA,L3.PY/L3.KF/H3.AE, L3.PY/L3.KF/H3.AQ L3.PY/L3.KF/H3.TAQ, P5A2_VHVL,A02_Rd4_0.6 nM_C06, A02_Rd4_0.6 nM_C09 A02_Rd4_6 nM_C16, A02_Rd4_6nM_C03, A02_Rd4_6 nM_C01 A02_Rd4_6 nM_C26 A02_Rd4_6 nM_C25, A02_Rd4_6nM_C22, A02_Rd4_6 nM_C19, A02_Rd4_0.6 nM_C03 A02_Rd4_6 nM_C07, A02_Rd4_6nM_C23, A02_Rd4_0.6 nM_C18, A02_Rd4_6 nM_C10 A02_Rd4_6 nM_C05,A02_Rd4_0.6 nM_C10, A02_Rd4_6 nM_C04, A02_Rd4_0.6 nM_C26A02_Rd4_0.6 nM_C13, A02_Rd4_0.6 nM_C01, A02_Rd4_6 nM_C08, P5C1_VHVL,C01_Rd4_6 nM_C24, C01_Rd4_6 nM_C26, C01_Rd4_6 nM_C10, C01_Rd4_0.6 nM_C27C01_Rd4_6 nM_C20, C01_Rd4_6 nM_C12, C01_Rd4_0.6 nM_C16, C01_Rd4_0.6nM_C09C01_Rd4_6 nM_C09, C01_Rd4_0.6 nM_C03, C01_Rd4_0.6 nM_C06, C01_Rd4_6nM_C04COMBO_Rd4_0.6 nM_C22, COMBO_Rd4_6 nM_C21, COMBO_Rd4_6 nM_C10,COMBO_Rd4_0.6 nM_C04, COMBO_Rd4_6 nM_C25, COMBO_Rd4_0.6 nM_C21,COMBO_Rd4_6 nM_C11, COMBO_Rd4_0.6 nM_C20, COMBO_Rd4_6 nM_C09,COMBO_Rd4_6 nM_C08, COMBO_Rd4_0.6 nM_C19, COMBO_Rd4_0.6 nM_C02,COMBO_Rd4_0.6 nM_C23, COMBO_Rd4_0.6 nM_C29, COMBO_Rd4_0.6 nM_C09,COMBO_Rd4_6 nM_C12, COMBO_Rd4_0.6 nM_C30, COMBO_Rd4_0.6 nM_C14,COMBO_Rd4_6 nM_C07, COMBO_Rd4_6 nM_C02, COMBO_Rd4_0.6 nM_C05,COMBO_Rd4_0.6 nM_C17, COMBO_Rd4_6 nM_C22, COMBO_Rd4_0.6 nM_C11,COMBO_Rd4_0.6 nM_C29, P4G4, or P1A11.In some embodiments, the present invention provides an antibody or anantigen binding fragment, which specifically binds to BCMA, wherein theantibody comprises a VH region comprising a sequence shown in SEQ ID NO:112; and/or a VL region comprising a sequence shown in SEQ ID NO: 38. Insome embodiments, the antibody comprises a light chain comprising thesequence EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 357) and a heavychain comprising the sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSLPYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 358).

In some embodiments, the present invention provides an antibody or anantigen binding fragment, which specifically bind to BCMA, wherein theantibody comprises a VH region comprising a sequence shown in SEQ ID NO:2, 32, 42, or 78; and/or a VL region comprising a sequence shown in SEQID NO: 6, 16, 43, or 85.

In some embodiments, the invention also provides CDR portions ofantibodies to BCMA antibodies based on CDR contact regions. CDR contactregions are regions of an antibody that imbue specificity to theantibody for an antigen. In general, CDR contact regions include theresidue positions in the CDRs and Vernier zones which are constrained inorder to maintain proper loop structure for the antibody to bind aspecific antigen. See, e.g., Makabe et al., J. Biol. Chem.,283:1156-1166, 2007. Determination of CDR contact regions is well withinthe skill of the art.

The binding affinity (K_(D)) of the BCMA antibody as described herein toBCMA (such as human BCMA (e.g., (SEQ ID NO: 353) can be about 0.002 nMto about 6500 nM. In some embodiments, the binding affinity is about anyof 6500 nm, 6000 nm, 5986 nm, 5567 nm, 5500 nm, 4500 nm, 4000 nm, 3500nm, 3000 nm, 2500 nm, 2134 nm, 2000 nm, 1500 nm, 1000 nm, 750 nm, 500nm, 400 nm, 300 nm, 250 nm, 200 nM, 193 nM, 100 nM, 90 nM, 50 nM, 45 nM,40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 19 nm, 18 nm, 17 nm, 16 nm, 15 nM, 10nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1nM, 0.5 nM, 0.3 nM, 0.1 nM, 0.01 nM, or 0.002 nM. In some embodiments,the binding affinity is less than about any of 6500 nm, 6000 nm, 5500nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, 1000 nm, 900 nm, 800 nm, 250 nM,200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM,5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

In some embodiments, the invention encompasses compositions, includingpharmaceutical compositions, comprising antibodies described herein ormade by the methods and having the characteristics described herein. Asused herein, compositions comprise one or more antibodies that bind toBCMA, and/or one or more polynucleotides comprising sequences encodingone or more these antibodies. These compositions may further comprisesuitable excipients, such as pharmaceutically acceptable excipientsincluding buffers, which are well known in the art.

The invention also provides methods of making any of these antibodies.The antibodies of this invention can be made by procedures known in theart. The polypeptides can be produced by proteolytic or otherdegradation of the antibodies, by recombinant methods (i.e., single orfusion polypeptides) as described above or by chemical synthesis.Polypeptides of the antibodies, especially shorter polypeptides up toabout 50 amino acids, are conveniently made by chemical synthesis.Methods of chemical synthesis are known in the art and are commerciallyavailable. For example, an antibody could be produced by an automatedpolypeptide synthesizer employing the solid phase method. See also, U.S.Pat. Nos. 5,807,715; 4,816,567; and 6,331,415.

The invention also encompasses fusion proteins comprising one or morefragments or regions from the antibodies of this invention. In oneembodiment, a fusion polypeptide is provided that comprises at least 10contiguous amino acids of the variable light chain region shown in SEQID NOs: 1, 4, 5, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 317, 81, 82, 84, 85, 86, 88, 89, 90, 91,93, 94, 96, 98, 100, 102, 103, 105, 107, 108, 109, 111, 113, 115, 116,117, 119, 121, 123, 124, 126, 128, 80, 315, 36, or 364, and/or at least10 amino acids of the variable heavy chain region shown in SEQ ID NOs:2, 3, 7, 8, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 37, 39, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 83,87, 92, 95, 97, 99, 101, 104, 106, 110, 112, 114, 118, 120, 122, 112,125, 127, 313, 314, 363, or 365. In other embodiments, a fusionpolypeptide is provided that comprises at least about 10, at least about15, at least about 20, at least about 25, or at least about 30contiguous amino acids of the variable light chain region and/or atleast about 10, at least about 15, at least about 20, at least about 25,or at least about 30 contiguous amino acids of the variable heavy chainregion. In another embodiment, the fusion polypeptide comprises a lightchain variable region and/or a heavy chain variable region, as shown inany of the sequence pairs selected from among SEQ ID NOs: 1 and 2, 1 and3, 4 and 2, 5 and 2, 6 and 2, 4 and 7, 4 and 8, 4 and 3, 9 and 8, 9 and3, 10 and 7, 10 and 8, 10 and 3, 11 and 7, 11 and 8, 11 and 3, 12 and 3,13 and 7, 13 and 8, 14 and 3, 15 and 2, 16 and 2, 17 and 2, 18 and 2, 19and 2, 20 and 2, 21 and 2, 22 and 2, 23 and 2, 16 and 24, 16 and 25, 16and 26, 16 and 27, 16 and 28, 16 and 29, 16 and 30, 16 and 31, 16 and 3,16 and 32, 16 and 2, 18 and 24, 18 and 25, 18 and 26, 18 and 27, 18 and28, 18 and 29, 18 and 30, 18 and 31, 18 and 3, 18 and 32, 19 and 24, 19and 25, 19 and 26, 19 and 27, 19 and 28, 19 and 29, 19 and 30, 19 and31, 19 and 3, 19 and 32, 20 and 24, 20 and 25, 20 and 26, 20 and 27, 20and 28, 20 and 29, 20 and 30, 20 and 31, 20 and 3, 20 and 32, 21 and 24,21 and 28, 21 and 31, 21 and 3, 21 and 32, 22 and 24, 22 and 25, 22 and26, 22 and 27, 22 and 28, 22 and 29, 22 and 30, 22 and 32, 23 and 25, 23and 26, 23 and 27, 23 and 29, 23 and 30, 23 and 31, 23 and 3, 23 and 32,34 and 33, 36 and 35, 38 and 37, 40 and 39, 41 and 33, 43 and 42, 45 and44, 47 and 46, 49 and 48, 51 and 50, 53 and 52, 55 and 54, 57 and 56, 59and 58, 61 and 60, 63 and 62, 65 and 64, 67 and 66, 69 and 68, 71 and70, 73 and 72, 75 and 74, 77 and 76, 79 and 78, 317 and 78, 79 and 78,81 and 78, 82 and 78, 84 and 83, 85 and 78, 86 and 78, 88 and 87, 89 and78, 90 and 78, 91 and 78, 93 and 92, 94 and 78, 96 and 95, 98 and 97, 38and 78, 102 and 101, 103 and 78, 105 and 104, 107 and 106, 108 and 78,109 and 78, 111 and 110, 38 and 112, 113 and 112, 115 and 114, 116 and76, 117 and 112, 119 and 118, 121 and 120, 123 and 122, 124 and 112, 126and 125, 128 and 127, 80 and 363, or 364 and 365. In another embodiment,the fusion polypeptide comprises one or more CDR(s). In still otherembodiments, the fusion polypeptide comprises CDR H3 (VH CDR3) and/orCDR L3 (VL CDR3). For purposes of this invention, a fusion proteincontains one or more antibodies and another amino acid sequence to whichit is not attached in the native molecule, for example, a heterologoussequence or a homologous sequence from another region. Exemplaryheterologous sequences include, but are not limited to a “tag” such as aFLAG tag or a 6His tag. Tags are well known in the art.

The invention also provides isolated polynucleotides encoding theantibodies of the invention, and vectors and host cells comprising thepolynucleotide.

In one embodiment, a polynucleotide comprises a sequence encoding theheavy chain and/or the light chain variable regions of antibodyP6E01/P6E01, P6E01/H3.AQ, L1.LGF/L3.KW/P6E01; L1.LGF/L3.NY/P6E01,L1.GDF/L3.NY/P6E01, L1.LGF/L3.KW/H3.AL, L1.LGF/L3.KW/H3.AP,L1.LGF/L3.KW/H3.AQ, L1.LGF/L3.PY/H3.AP, L1.LGF/L3.PY/H3.AQ,L1.LGF/L3.NY/H3.AL, L1.LGF/L3.NY/H3.AP, L1.LGF/L3.NY/H3.AQ,L1.GDF/L3.KW/H3.AL, L1.GDF/L3.KW/H3.AP, L1.GDF/L3.KW/H3.AQ,L1.GDF/L3.PY/H3.AQ, L1.GDF/L3.NY/H3.AL, L1.GDF/L3.NY/H3.AP,L1.GDF/L3.NY/H3.AQ, L3.KW/P6E01, L3.PY/P6E01, L3.NY/P6E01,

L3.PY/L1.PS/P6E01, L3.PY/L1.AH/P6E01, L3.PY/L1.FF/P6E01,L3.PY/L1.PH/P6E01, L3.PY/L3.KY/P6E01, L3.PY/L3.KF/P6E01, L3.PY/H2.QR,L3.PY/H2.DY, L3.PY/H2.YQ, L3.PY/H2.LT, L3.PY/H2.HA, L3.PY/H2.QL,L3.PY/H3.YA, L3.PY/H3.AE, L3.PY/H3.AQ, L3.PY/H3.TAQ, L3.PY/P6E01,L3.PY/L1.PS/H2.QR, L3.PY/L1.PS/H2.DY, L3.PY/L1.PS/H2.YQ,L3.PY/L1.PS/H2.LT, L3.PY/L1.PS/H2.HA, L3.PY/L1.PS/H2.QL,L3.PY/L1.PS/H3.YA, L3.PY/L1.PS/H3.AE, L3.PY/L1.PS/H3.AQ,L3.PY/L1.PS/H3.TAQ, L3.PY/L1.AH/H2.QR, L3.PY/L1.AH/H2.DY,L3.PY/L1.AH/H2.YQ, L3.PY/L1.AH/H2.LT, L3.PY/L1.AH/H2.HA,L3.PY/L1.AH/H2.QL, L3.PY/L1.AH/H3.YA, L3.PY/L1.AH/H3.AE,L3.PY/L1.AH/H3.AQ, L3.PY/L1.AH/H3.TAQ, L3.PY/L1.FF/H2.QR,L3.PY/L1.FF/H2.DY, L3.PY/L1.FF/H2.YQ, L3.PY/L1.FF/H2.LT,L3.PY/L1.FF/H2.HA, L3.PY/L1.FF/H2.QL, L3.PY/L1.FF/H3.YA,L3.PY/L1.FF/H3.AE, L3.PY/L1.FF/H3.AQ, L3.PY/L1.FF/H3.TAQ,L3.PY/L1.PH/H2.QR, L3.PY/L1.PH/H2.HA, L3.PY/L1.PH/H3.AE,L3.PY/L1.PH/H3.AQ, L3.PY/L1.PH/H3.TAQ, L3.PY/L3.KY/H2.QR,L3.PY/L3.KY/H2.DY, L3.PY/L3.KY/H2.YQ L3.PY/L3.KY/H2.LT,L3.PY/L3.KY/H2.HA, L3.PY/L3.KY/H2.QL, L3.PY/L3.KY/H3.YAL3.PY/L3.KY/H3.TAQ, L3.PY/L3.KF/H2.DY, L3.PY/L3.KF/H2.YQ,L3.PY/L3.KF/H2.LT L3.PY/L3.KF/H2.QL, L3.PY/L3.KF/H3.YA,L3.PY/L3.KF/H3.AE, L3.PY/L3.KF/H3.AQ L3.PY/L3.KF/H3.TAQ, P5A2_VHVL,A02_Rd4_0.6 nM_C06, A02_Rd4_0.6 nM_C09 A02_Rd4_6 nM_C16, A02_Rd4_6nM_C03, A02_Rd4_6 nM_C01, A02_Rd4_6 nM_C26 A02_Rd4_6 nM_C25, A02_Rd4_6nM_C22, A02_Rd4_6 nM_C19, A02_Rd4_0.6 nM_C03 A02_Rd4_6 nM_C07, A02_Rd4_6nM_C23, A02_Rd4_0.6 nM_C1 A02_Rd4_6 nM_C10 A02_Rd4_6 nM_C05, A02_Rd4_0.6nM_C10, A02_Rd4_6 nM_C04, A02_Rd4_0.6 nM_C26A02_Rd4_0.6 nM_C13, A02_Rd4_0.6 nM_C01, A02_Rd4_6 nM_C08, P5C1_VHVL,C01_Rd4_6 nM_C24, C01_Rd4_6 nM_C26, C01_Rd4_6 nM_C10, C01_Rd4_0.6 nM_C27C01_Rd4_6 nM_C20, C01_Rd4_6 nM_C12, C01_Rd4_0.6 nM_C16, C01_Rd4_0.6nM_C09C01_Rd4_6 nM_C09, C01_Rd4_0.6 nM_C03, C01_Rd4_0.6 nM_C06, C01_Rd4_6nM_C04COMBO_Rd4_0.6 nM_C22, COMBO_Rd4_6 nM_C21, COMBO_Rd4_6 nM_C10,COMBO_Rd4_0.6 nM_C04, COMBO_Rd4_6 nM_C25, COMBO_Rd4_0.6 nM_C21,COMBO_Rd4_6 nM_C11, COMBO_Rd4_0.6 nM_C20, COMBO_Rd4_6 nM_C09,COMBO_Rd4_6 nM_C08, COMBO_Rd4_0.6 nM_C19, COMBO_Rd4_0.6 nM_C02,COMBO_Rd4_0.6 nM_C23, COMBO_Rd4_0.6 nM_C29, COMBO_Rd4_0.6 nM_C09,COMBO_Rd4_6 nM_C12, COMBO_Rd4_0.6 nM_C30, COMBO_Rd4_0.6 nM_C14,COMBO_Rd4_6 nM_C07, COMBO_Rd4_6 nM_C02, COMBO_Rd4_0.6 nM_C05,COMBO_Rd4_0.6 nM_C17, COMBO_Rd4_6 nM_C22, COMBO_Rd4_0.6 nM_C11,COMBO_Rd4_0.6 nM_C29, P4G4, or P1A11. The sequence encoding the antibodyof interest may be maintained in a vector in a host cell and the hostcell can then be expanded and frozen for future use. Vectors (includingexpression vectors) and host cells are further described herein.

The invention also encompasses scFv of antibodies of this invention.Single chain variable region fragments are made by linking light and/orheavy chain variable regions by using a short linking peptide (Bird etal., Science 242:423-426, 1988). An example of a linking peptide is(GGGGS)₃ (SEQ ID NO: 498), which bridges approximately 3.5 nm betweenthe carboxy terminus of one variable region and the amino terminus ofthe other variable region. Linkers of other sequences have been designedand used (Bird et al., 1988, supra). Linkers should be short, flexiblepolypeptides and preferably comprised of less than about 20 amino acidresidues. Linkers can in turn be modified for additional functions, suchas attachment of drugs or attachment to solid supports. The single chainvariants can be produced either recombinantly or synthetically. Forsynthetic production of scFv, an automated synthesizer can be used. Forrecombinant production of scFv, a suitable plasmid containingpolynucleotide that encodes the scFv can be introduced into a suitablehost cell, either eukaryotic, such as yeast, plant, insect or mammaliancells, or prokaryotic, such as E. coli. Polynucleotides encoding thescFv of interest can be made by routine manipulations such as ligationof polynucleotides. The resultant scFv can be isolated using standardprotein purification techniques known in the art.

Other forms of single chain antibodies, such as diabodies or minibodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich heavy chain variable (VH) and light chain variable (VL) domainsare expressed on a single polypeptide chain, but using a linker that istoo short to allow for pairing between the two domains on the samechain, thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (see e.g.,Holliger, P., et al., Proc. Natl. Acad Sci. USA 90:6444-6448, 1993;Poljak, R. J., et al., Structure 2:1121-1123, 1994). Minibody includesthe VL and VH domains of a native antibody fused to the hinge region andCH3 domain of the immunoglobulin molecule. See, e.g., U.S. Pat. No.5,837,821.

In another aspect, the invention provides compositions (such as apharmaceutical compositions) comprising any of the polynucleotides ofthe invention. In some embodiments, the composition comprises anexpression vector comprising a polynucleotide encoding any of theantibodies described herein. In still other embodiments, the compositioncomprises either or both of the polynucleotides shown in SEQ ID NO: 486and SEQ ID NO: 485 below:

COMBO_Rd4_0.6nM_C29 heavy chain variable region (SEQ ID NO: 486)AAGTCCAACTCCTCGAATCCGGTGGCGGCCTTGTCCAGCCTGGAGGTTCCTTGCGCCTGTCATGTGCCGCCAGCGGATTCACCTTCTCGTCCTACCCGATGTCGTGGGTCCGCCAGGCTCCGGGAAAGGGCCTGGAATGGGTGTCAGCCATCGGAGGATCGGGGGGCTCCCTGCCCTACGCCGATATCGTGAAGGGAAGGTTCACCATTAGCCGGGACAACTCCAAGAACACTCTGTACCTCCAAATGAACAGCCTGAGAGCGGAGGACACCGCAGTGTACTATTGCGCCCGGTACTGGCCAATGGACATCTGGGGCCAGGGGACTCTGGTCACCGTCTCCTCA COMBO_Rd4_0.6nM_C29 lightchain variable region (SEQ ID NO: 485)GAGATCGTGCTGACTCAGTCCCCTGGAACCCTGTCCCTGTCACCTGGCGAAAGAGCTACCTTGTCCTGTCGCGCATCACAATCCGTGTCGTCGAGCTATCTCGCGTGGTACCAGCAGAAGCCCGGACAGGCCCCAAGGCTGCTTATGTACGACGCCTCCATCCGGGCCACTGGTATCCCCGACCGCTTCTCGGGCTCCGGAAGCGGCACCGACTTCACCCTGACTATTTCCCGGCTCGAACCGGAGGATTTCGCCGTGTACTACTGCCAACAGTACCAGAGCTGGCCGCTGACGTTTGGGCAGGGGACCAAGGTCGAAATCAAA

In other embodiments, the composition comprises either or both of thepolynucleotides shown in SEQ ID NO: 488 and SEQ ID NO: 487 below:

L3.PY/H3TAQ heavy chain variable region (SEQ ID NO: 488)GAAGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCGGCAGCTACGCTATGACCTGGGTGCGCCAGGCCCCTGGCAAAGGACTGGAATGGGTGTCCGCCATCTCTGGCAGCGGCGGCAATACCTTCTACGCCGAGAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTACACGGGTGTCCCCTATCGCCGCGCAGATGGATTATTGGGGCCAGGGCACTCTGGTCACCGT CTCCTCA L3.PY/H3TAQheavy chain variable region (SEQ ID NO: 487)GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCAGGCGAAAGAGCCACCCTGTCCTGCAGAGCCAGCCAGAGCGTGTCCAGCAGCTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCTCCCCGGCTGCTGATCTATGGCGCCTCTTCTAGAGCCACCGGCATCCCCGATAGATTCAGCGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCACTACCCTTATCCCCCCAGCTTCACATTTGGCCAGGGCACCAAGGTGGAGATCAAA

In still other embodiments, the composition comprises either or both ofthe polynucleotides shown in SEQ ID NO: 490 and SEQ ID NO: 489 below:

A02_Rd4_0.6nM_C01 heavy chain variable region (SEQ ID NO: 490)GAAGTTCAATTATTGGAATCTGGTGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCTATTACTGCGTCTGGTGGTTCTACTTACTATGCCGATGTGGTTAAGGGTAGATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTGCTAGATACTGGCCAATGTCGTTGTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA A02_Rd4_0.6nM_C01 lightchain variable region (SEQ ID NO: 489)GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGAAAGAGCTACTTTGTCTTGTAGAGCTTCTCAATCCGTTTCCGCGTATTATTTGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATGTACGATGCTTCTATTAGAGCCACCGGTATTCCAGATAGATTTTCTGGTTCTGGTTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATTTCGCTGTTTACTACTGTCAACAATATGAGCGTTGGCCATTGACTTTTGGTCAAGGTACAAAGGTTGAAATCAAACGTGAG

In other embodiments, the composition comprises either or both of thepolynucleotides shown in SEQ ID NO: 492 and SEQ ID NO: 491 below:

A02_Rd4_0.6nM_C16 heavy chain variable region (SEQ ID NO: 492)GAAGTTCAATTATTGGAATCTGGTGGAGGACTGGTGCAGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGTAAAGGTTTGGAATGGGTTTCTGCTATTTCTGATTTTGGTGGTTCTACTTACTATGCCGATATCGTTAAGGGTAGATTCACCATTTCTAGAGACAACTCTAAGAACACCTTGTACTTGCAAATGAACTCCTTGAGAGCTGAAGATACTGCTGTTTATTACTGTGCTAGATACTGGCCAATGGATATTTGGGGTCAAGGTACTCTGGTCACCGTCTCCTCA A02_Rd4_0.6nM_C16 lightchain variable region (SEQ ID NO: 491)GAGATCGTGCTGACACAGAGCCCTGGCACCCTGAGCCTGTCTCCTGGTGAAAGAGCTACTTTGTCTTGTAGAGCTTCTCAATCCGTTTCCGATCTGTATTTGGCTTGGTATCAACAAAAACCAGGTCAAGCTCCAAGATTATTGATGTACGATGCTTCTATTAGAGCCACCGGTATTCCAGATAGATTTTCTGGTTCTGGTTCCGGTACTGATTTCACTTTGACTATCTCTAGATTGGAACCAGAAGATTTCGCTGTTTACTACTGTCAACAATATCAGACTTGGCCATTGACTTTTGGTCAAGGTACAAAGGTTGAAATCAAACGTGAG.

Expression vectors, and administration of polynucleotide compositionsare further described herein.

In another aspect, the invention provides a method of making any of thepolynucleotides described herein.

Polynucleotides complementary to any such sequences are also encompassedby the present invention. Polynucleotides may be single-stranded (codingor antisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. RNA molecules include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or non-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an antibody or a portion thereof) or may comprisea variant of such a sequence. Polynucleotide variants contain one ormore substitutions, additions, deletions and/or insertions such that theimmunoreactivity of the encoded polypeptide is not diminished, relativeto a native immunoreactive molecule. The effect on the immunoreactivityof the encoded polypeptide may generally be assessed as describedherein. Variants preferably exhibit at least about 70% identity, morepreferably, at least about 80% identity, yet more preferably, at leastabout 90% identity, and most preferably, at least about 95% identity toa polynucleotide sequence that encodes a native antibody or a portionthereof.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, or 40 to about 50, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O., 1978, A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e. the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a nativeantibody (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

The polynucleotides of this invention can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al., 1989.

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., 1989, supra, for example.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a polynucleotide according to the invention. It is impliedthat an expression vector must be replicable in the host cells either asepisomes or as an integral part of the chromosomal DNA. Suitableexpression vectors include but are not limited to plasmids, viralvectors, including adenoviruses, adeno-associated viruses, retroviruses,cosmids, and expression vector(s) disclosed in PCT Publication No. WO87/04462. Vector components may generally include, but are not limitedto, one or more of the following: a signal sequence; an origin ofreplication; one or more marker genes; suitable transcriptionalcontrolling elements (such as promoters, enhancers and terminator). Forexpression (i.e., translation), one or more translational controllingelements are also usually required, such as ribosome binding sites,translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

The invention also provides host cells comprising any of thepolynucleotides described herein. Any host cells capable ofover-expressing heterologous DNAs can be used for the purpose ofisolating the genes encoding the antibody, polypeptide or protein ofinterest. Non-limiting examples of mammalian host cells include but notlimited to COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such asE. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; orK. lactis). Preferably, the host cells express the cDNAs at a level ofabout 5 fold higher, more preferably, 10 fold higher, even morepreferably, 20 fold higher than that of the corresponding endogenousantibody or protein of interest, if present, in the host cells.Screening the host cells for a specific binding to BCMA or an BCMAdomain (e.g., domains 1-4) is effected by an immunoassay or FACS. A celloverexpressing the antibody or protein of interest can be identified.

Representative materials of the present invention were deposited in theAmerican Type Culture Collection (ATCC) on Apr. 15, 2015. Vector havingATCC Accession No. PTA-122094 is a polynucleotide encoding a humanizedBCMA antibody heavy chain variable region, and vector having ATCCAccession No. PTA-122093 is a polynucleotide encoding a humanized BCMAantibody light chain variable region. The deposits were made under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purpose of Patent Procedure andRegulations thereunder (Budapest Treaty). This assures maintenance of aviable culture of the deposit for 30 years from the date of deposit. Thedeposit will be made available by ATCC under the terms of the BudapestTreaty, and subject to an agreement between Pfizer, Inc. and ATCC, whichassures permanent and unrestricted availability of the progeny of theculture of the deposit to the public upon issuance of the pertinent U.S.patent or upon laying open to the public of any U.S. or foreign patentapplication, whichever comes first, and assures availability of theprogeny to one determined by the U.S. Commissioner of Patents andTrademarks to be entitled thereto according to 35 U.S.C. Section 122 andthe Commissioner's rules pursuant thereto (including 37 C.F.R. Section1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

BCMA Antibody Conjugates

The present invention also provides a conjugate (or immunoconjugate) ofthe BCMA antibody as described herein, or of the antigen bindingfragment thereof, wherein the antibody or the antigen binding fragmentis conjugated to an agent (e.g., a cytotoxic agent) for targetedimmunotherapy (e.g., antibody-drug conjugates) either directly orindirectly via a linker. For example, a cytotoxic agent can be linked orconjugated to the BCMA antibody or the antigen binding fragment thereofas described herein for targeted local delivery of the cytotoxic agentmoiety to tumors (e.g., BCMA expressing tumor).

Methods for conjugating cytotoxic agent or other therapeutic agents toantibodies have been described in various publications. For example,chemical modification can be made in the antibodies either throughlysine side chain amines or through cysteine sulfhydryl groups activatedby reducing interchain disulfide bonds for the conjugation reaction tooccur. See, e.g., Tanaka et al., FEBS Letters 579:2092-2096, 2005, andGentle et al., Bioconjugate Chem. 15:658-663, 2004. Reactive cysteineresidues engineered at specific sites of antibodies for specific drugconjugation with defined stoichiometry have also been described. See,e.g., Junutula et al., Nature Biotechnology, 26:925-932, 2008.Conjugation using an acyl donor glutamine-containing tag or anendogenous glutamine made reactive (i.e., the ability to form a covalentbond as an acyl donor) by polypeptide engineering in the presence oftransglutaminase and an amine (e.g., a cytotoxic agent comprising orattached to a reactive amine) is also described in internationalapplications WO2012/059882 and WO2015015448.

In some embodiments, the BCMA antibody or the conjugate as describedherein comprises an acyl donor glutamine-containing tag engineered at aspecific site of the antibody (e.g., a carboxyl terminus, an aminoterminus, or at another site in the BCMA antibody). In some embodiments,the tag comprises an amino acid glutamine (Q) or an amino acid sequenceLQG, LLQGG (SEQ ID NO:318), LLQG (SEQ ID NO:454), LSLSQG (SEQ ID NO:455), GGGLLQGG (SEQ ID NO: 456), GLLQG (SEQ ID NO: 457), LLQ, GSPLAQSHGG(SEQ ID NO: 458), GLLQGGG (SEQ ID NO: 459), GLLQGG (SEQ ID NO: 460),GLLQ (SEQ ID NO: 461), LLQLLQGA (SEQ ID NO: 462), LLQGA (SEQ ID NO:463), LLQYQGA (SEQ ID NO: 464), LLQGSG (SEQ ID NO: 465), LLQYQG (SEQ IDNO: 466), LLQLLQG (SEQ ID NO: 467), SLLQG (SEQ ID NO: 468), LLQLQ (SEQID NO: 469), LLQLLQ (SEQ ID NO: 470), LLQGR (SEQ ID NO: 471), LLQGPP(SEQ ID NO: 472), LLQGPA (SEQ ID NO: 473), GGLLQGPP (SEQ ID NO: 474),GGLLQGA (SEQ ID NO: 475), LLQGPGK (SEQ ID NO: 476), LLQGPG (SEQ ID NO:477), LLQGP (SEQ ID NO: 478), LLQP (SEQ ID NO: 479), LLQPGK (SEQ ID NO:480), LLQAPGK (SEQ ID NO: 481), LLQGAPG (SEQ ID NO: 482), LLQGAP (SEQ IDNO: 483), and LLQLQG (SEQ ID NO: 484).

In some embodiments, the BCMA antibody or the conjugate as describedherein comprises an acyl donor glutamine-containing tag engineered at aspecific site of the antibody, wherein the tag comprises an amino acidsequence GGLLQGPP (SEQ ID NO: 474) or GGLLQGA (SEQ ID NO: 475)engineered at the light chain carboxyl terminus of the BCMA antibody. Insome embodiments, the BCMA antibody or the conjugate as described hereincomprises an acyl donor glutamine-containing tag engineered at aspecific site of the antibody, wherein the tag comprises an amino acidsequence LLQG (SEQ ID NO: 454) engineered after residue T135 in theheavy chain of the BCMA antibody. In other embodiments, the BCMAantibody or the conjugate as described herein comprises an acyl donorglutamine-containing tag engineered at a specific site of the antibody,wherein the tag comprises an amino acid sequence LLQGA (SEQ ID NO: 463)or LLQGPP (SEQ ID NO: 472) engineered at the heavy chain carboxylterminus of the BCMA antibody and wherein the lysine residue at theheavy chain carboxyl terminus is deleted. In some embodiments, the BCMAantibody or the conjugate as described herein comprises an amino acidsubstitution at position 297 of the BCMA antibody (EU numbering scheme).For example, the amino acid asparagine (N) can be substituted withglutamine (Q) or alanine (A) at position 297 of the BCMA antibody.

Also provided is an isolated antibody comprising an acyl donorglutamine-containing tag and an amino acid modification at position 222,340, or 370 of the antibody (EU numbering scheme) wherein themodification is an amino acid deletion, insertion, substitution,mutation, or any combination thereof. Accordingly, in some embodiments,provided is the BCMA antibody or the conjugate as described hereincomprising the acyl donor glutamine-containing tag (e.g., Q, LQG, LLQGG(SEQ ID NO:318), LLQG (SEQ ID NO:454), LSLSQG (SEQ ID NO: 455), GGGLLQGG(SEQ ID NO: 456), GLLQG (SEQ ID NO: 457), LLQ, GSPLAQSHGG (SEQ ID NO:458), GLLQGGG (SEQ ID NO: 459), GLLQGG (SEQ ID NO: 460), GLLQ (SEQ IDNO: 461), LLQLLQGA (SEQ ID NO: 462), LLQGA (SEQ ID NO: 463), LLQYQGA(SEQ ID NO: 464), LLQGSG (SEQ ID NO: 465), LLQYQG (SEQ ID NO: 466),LLQLLQG (SEQ ID NO: 467), SLLQG (SEQ ID NO: 468), LLQLQ (SEQ ID NO:469), LLQLLQ (SEQ ID NO: 470), LLQGR (SEQ ID NO: 471), LLQGPP (SEQ IDNO: 472), LLQGPA (SEQ ID NO: 473), GGLLQGPP (SEQ ID NO: 474), GGLLQGA(SEQ ID NO: 475), LLQGPGK (SEQ ID NO: 476), LLQGPG (SEQ ID NO: 477),LLQGP (SEQ ID NO: 478), LLQP (SEQ ID NO: 479), LLQPGK (SEQ ID NO: 480),LLQAPGK (SEQ ID NO: 481), LLQGAPG (SEQ ID NO: 482), LLQGAP (SEQ ID NO:483), and LLQLQG (SEQ ID NO: 484)) conjugated at a specific site (e.g.,at a carboxyl terminus of the heavy or light chain, residue T135 in theantibody heavy chain, or at another site) of the BCMA antibody and anamino acid modification at position 222, 340, or 370 of the antibody (EUnumbering scheme). In some embodiments, the amino acid modification is asubstitution from lysine to arginine (e.g., K222R, K340R, or K370R).

In some embodiments, the BCMA antibody or the conjugate as describedherein comprises an acyl donor glutamine-containing tag comprising thesequence GGLLQGPP (SEQ ID NO: 474) engineered at the C-terminus of theBCMA antibody light chain and an amino acid substitution from lysine toarginine at position 222 of the antibody (EU numbering scheme). In someembodiments, the BCMA antibody or the conjugate as described hereincomprises an acyl donor glutamine-containing tag comprising the sequenceGGLLQGA (SEQ ID NO: 475) engineered at the C-terminus of the BCMAantibody light chain and an amino acid substitution from lysine toarginine at position 222 of the antibody (EU numbering scheme). In someembodiments, the BCMA antibody or the conjugate as described hereincomprises an acyl donor glutamine-containing tag comprising the sequenceLLQGA (SEQ ID NO: 463) engineered at the C-terminus of the BCMA antibodyheavy chain and an amino acid substitution from lysine to arginine atposition 222 of the antibody (EU numbering scheme), wherein the lysineresidue at the heavy chain carboxyl terminus is deleted. In someembodiments, the BCMA antibody or the conjugate as described hereincomprises an acyl donor glutamine-containing tag comprising the sequenceLLQG (SEQ ID NO: 454) engineered after residue T135 in the heavy chainof the BCMA antibody and an amino acid substitution from lysine toarginine at position 222 of the antibody (EU numbering scheme).

In some embodiments, the BCMA antibody or the conjugate as describedherein comprises an acyl donor glutamine-containing tag comprising aglutamine engineered at position 297 or an amino acid substitution atposition 297 from asparagine (N) to another amino acid in the BCMAantibody and an amino acid substitution from lysine to arginine atposition 222 of the antibody (EU numbering scheme). For example, in someembodiments, the BCMA antibody or the conjugate as described hereincomprises an acyl donor glutamine-containing tag comprising the sequenceGGLLQGPP (SEQ ID NO: 474) engineered at the C-terminus of the BCMAantibody light chain, an amino acid substitution at position 297 of theBCMA antibody from asparagine (N) to glutamine (Q), and an amino acidsubstitution from lysine to arginine at position 222 of the antibody (EUnumbering scheme). In some embodiments, the BCMA antibody or theconjugate as described herein comprises an acyl donorglutamine-containing tag comprising the sequence LLQG (SEQ ID NO: 454)engineered after residue T135 in the heavy chain of the BCMA antibody,an amino acid substitution at position 297 of the BCMA antibody fromasparagine (N) to alanine (A), and an amino acid substitution fromlysine to arginine at position 222 of the antibody (EU numberingscheme).

The agents that can be conjugated to the BCMA antibodies or the antigenbinding fragments of the present invention include, but are not limitedto, cytotoxic agents, immunomodulating agents, imaging agents,therapeutic proteins, biopolymers, or oligonucleotides.

Examples of a cytotoxic agent include, but are not limited to,anthracycline, an auristatin, a dolastatin, a combretastatin, aduocarmycin, a pyrrolobenzodiazepine dimer, an indolino-benzodiazepinedimer, an enediyne, a geldanamycin, a maytansine, a puromycin, a taxane,a vinca alkaloid, a camptothecin, a tubulysin, a hemiasterlin, aspliceostatin, a pladienolide, and stereoisomers, isosteres, analogs, orderivatives thereof.

The anthracyclines are derived from bacteria Strepomyces and have beenused to treat a wide range of cancers, such as leukemias, lymphomas,breast, uterine, ovarian, and lung cancers. Exemplary anthracyclinesinclude, but are not limited to, daunorubicin, doxorubicin (i.e.,adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.

Dolastatins and their peptidic analogs and derivatives, auristatins, arehighly potent antimitotic agents that have been shown to have anticancerand antifungal activity. See, e.g., U.S. Pat. No. 5,663,149 and Pettitet al., Antimicrob. Agents Chemother. 42:2961-2965, 1998. Exemplarydolastatins and auristatins include, but are not limited to, dolastatin10, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), MMAD(Monomethyl Auristatin D or monomethyl dolastatin 10), MMAF (MonomethylAuristatin F orN-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE(Monomethyl Auristatin E orN-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine),5-benzoylvaleric acid-AE ester (AEVB), and other novel auristatins (suchas the ones described in U.S. Publication No. 2013/0129753). In someembodiments, the auristatin is 0101(2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide)having the following structure:

In some embodiments, the auristatin is 3377(N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxyl-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide)having the following structure:

In some embodiments, the auristatin is 0131-OMe(N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methylL-valinamide)having the following structure:

In other embodiments, the auristatin is 0131(2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide)having the following structure:

In other embodiments, the auristatin is 0121(2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide)having the following structure:

Camptothecin is a cytotoxic quinoline alkaloid which inhibits the enzymetopoisomerase I. Examples of camptothecin and its derivatives include,but are not limited to, topotecan and irinotecan, and their metabolites,such as SN-38.

Combretastatins are natural phenols with vascular disruption propertiesin tumors. Exemplary combretastatins and their derivatives include, butare not limited to, combretastatin A-4 (CA-4) and ombrabulin.

Duocarmycin and CC-1065 are DNA alkylating agents with cytotoxicpotency. See Boger and Johnson, PNAS 92:3642-3649 (1995). Exemplaryduocarmycin and CC-1065 include, but are not limited to, (+)-duocarmycinA and (+)-duocarmycin SA, (+)-CC-1065, and the compounds as disclosed inthe international application PCT/IB2015/050280 including, but notlimited to,N˜2˜-acetyl-L-lysyl-L-valyl-N˜5˜-carbamoyl-N-[4-({[(2-{[({(1S)-1-(chloromethyl)-3-[(5-{[(1S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl]carbonyl}thiophen-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamidehaving the structure:

N˜2˜-acetyl-L-lysyl-L-valyl-N˜5˜-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(3-{[(1S)-1-(chloromethyl)-8-methyl-5-(phosphonooxy)-1,6-dihydropyrrolo[3,2-e]indol-3(2H)-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamidehaving the structure:

N˜2˜-acetyl-L-lysyl-L-valyl-N˜5˜-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(4-{[(1S)-1-(chloromethyl)-8-methyl-5-(phosphonooxy)-1,6-dihydropyrrolo[3,2-e]indol-3(2H)-yl]carbonyl}pentacyclo[4.2.0.0˜2,5˜.0˜3,8˜.0˜4,7˜]oct-1-yl)carbonyl]-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamidehaving the structure:

Enediynes are a class of anti-tumor bacterial products characterized byeither nine- and ten-membered rings or the presence of a cyclic systemof conjugated triple-double-triple bonds. Exemplary enediynes include,but are not limited to, calicheamicin, esperamicin, uncialamicin,dynemicin, and their derivatives.

Geldanamycins are benzoquinone ansamycin antibiotic that bind to Hsp90(Heat Shock Protein 90) and have been used antitumor drugs. Exemplarygeldanamycins include, but are not limited to, 17-AAG(17-N-Allylamino-17-Demethoxygeldanamycin) and 17-DMAG(17-Dimethylaminoethylamino-17-demethoxygeldanamycin).

Hemiasterlin and its analogues (e.g., HTI-286) bind to the tubulin,disrupt normal microtubule dynamics, and, at stoichiometric amounts,depolymerize microtubules.

Maytansines or their derivatives maytansinoids inhibit cellproliferation by inhibiting the microtubules formation during mitosisthrough inhibition of polymerization of tubulin. See Remillard et al.,Science 189:1002-1005, 1975. Exemplary maytansines and maytansinoidsinclude, but are not limited to, mertansine (DM1) and its derivatives aswell as ansamitocin.

Pyrrolobenzodiazepine dimers (PBDs) and indolino-benzodiazepine dimers(IGNs) are anti-tumor agents that contain one or more immine functionalgroups, or their equivalents, that bind to duplex DNA. PBD and IGNmolecules are based on the natural product athramycin, and interact withDNA in a sequence-selective manner, with a preference forpurine-guanine-purine sequences. Exemplary PBDs and their analogsinclude, but are not limited to, SJG-136.

Spliceostatins and pladienolides are anti-tumor compounds which inhibitsplicing and interacts with spliceosome, SF3b. Examples ofspliceostatins include, but are not limited to, spliceostatin A,FR901464, and(2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-7-(2-hydrazinyl-2-oxoethyl)-4-hydroxy-1,6-dioxaspiro[2.5]oct-5-yl]-3-methylpenta-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-ylacetate having the structure of

Examples of pladienolides include, but are not limited to, PladienolideB, Pladienolide D, or E7107.

Taxanes are diterpenes that act as anti-tubulin agents or mitoticinhibitors. Exemplary taxanes include, but are not limited to,paclitaxel (e.g., TAXOL®) and docetaxel (TAXOTERE®).

Tubulysins are natural products isolated from a strain of myxobacteriathat has been shown to depolymerize microtubules and induce mitoticarrest. Exemplary tubulysins include, but are not limited to, tubulysinA, tubulysin B, and tubulysin D.

Vinca alkyloids are also anti-tubulin agents. Exemplary vinca alkyloidsinclude, but are not limited to, vincristine, vinblastine, vindesine,and vinorelbine.

Accordingly, in some embodiments, the cytotoxic agent is selected fromthe group consisting of MMAD (Monomethyl Auristatin D), 0101(2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide),3377(N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxyl-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide),0131(2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide),0131-OMe(N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methylL-valinamide),0121(2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide),and(2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-7-(2-hydrazinyl-2-oxoethyl)-4-hydroxy-1,6-dioxaspiro[2.5]oct-5-yl]-3-methylpenta-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-ylacetate.

In some embodiments, the agent is an immunomodulating agent. Examples ofan immunomodulating agent include, but are not limited to, gancyclovier,etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin,cyclophosphamide, azathioprine, mycophenolgate mofetil, methotrextrate,glucocorticoid and its analogs, cytokines, stem cell growth factors,lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors,interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10,IL-12, IL-18, and IL-21), colony stimulating factors (e.g.,granulocyte-colony stimulating factor (G-CSF) and granulocytemacrophage-colony stimulating factor (GM-CSF)), interferons (e.g.,interferons-α, -β and -γ), the stem cell growth factor designated “S 1factor,” erythropoietin and thrombopoietin, or a combination thereof.

In some embodiments, the agent moiety is an imaging agent (e.g., afluorophore or a chelator), such as fluorescein, rhodamine, lanthanidephosphors, and their derivatives thereof, or a radioisotope bound to achelator. Examples of fluorophores include, but are not limited to,fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite(FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, AlexaFluor® (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568,594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine(TAMRA) (e.g., 5,-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine(SR) (e.g., SR101). Examples of chelators include, but are not limitedto, 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA),1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (deferoxamine),diethylenetriaminepentaacetic acid (DTPA), and1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (BAPTA).

Examples of fluorophores include, but are not limited to, fluoresceinisothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g.,5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (e.g.,Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633,647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g.,5,-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g.,SR101).

In some embodiments, therapeutic or diagnostic radioisotopes or otherlabels (e.g., PET or SPECT labels) can be incorporated in the agent forconjugation to the BCMA antibodies or the antigen binding fragments asdescribed herein. Examples of a radioisotope or other labels include,but are not limited to, ³H, ¹¹C, ¹³N, ¹⁴C, ¹⁵N, ¹⁵O, ³⁵S, ¹⁸F, ³²P, ³³P,⁴⁷Sc, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Se, ⁷⁶Br,⁷⁷Br, ⁸⁶Y, ⁸⁹Zr, ⁹⁰Y, ⁹⁴Tc, ⁹⁵Ru, ⁹⁷Ru, ⁹⁹Tc, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁵Ru,¹⁰⁷Hg, ¹⁰⁹Pd, ¹¹¹Ag, ¹¹¹In, ¹¹³In, ¹²¹Te, ¹²²Te, ¹²³I, ¹²⁴I, ¹²⁵I,¹²⁵Te, ¹²⁶I, ¹³¹I, ¹³¹In, ¹³³I, ¹⁴²Pr, ¹⁴³Pr, ¹⁵³Pb, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁵Tm, ¹⁶⁶Dy, ¹⁶⁶H, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁶⁹Yb, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re,¹⁹⁷Pt, ¹⁹⁸Au, ¹⁹⁹Au, ²⁰¹Tl, ²⁰³Hg, ²¹¹At, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra,²²⁴Ac, or ²²⁵Ac.

In some embodiments, the agent is a therapeutic protein including, butis not limited to, a toxin, a hormone, an enzyme, and a growth factor.

Examples of a toxin protein (or polypeptide) include, but are notlimited to, dipththeria (e.g., diphtheria A chain), Pseudomonas exotoxinand endotoxin, ricin (e.g., ricin A chain), abrin (e.g., abrin A chain),modeccin (e.g., modeccin A chain), alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalisinhibitor, mitogellin, restrictocin, phenomycin, enomycin,tricothecenes, inhibitor cystine knot (ICK) peptides (e.g.,ceratotoxins), and conotoxin (e.g., KIIIA or SmIIIa).

In some embodiments, the agent is a biocompatible polymer. The BCMAantibodies or the antigen binding fragments as described herein can beconjugated to the biocompatible polymer to increase serum half-life andbioactivity, and/or to extend in vivo half-lives. Examples ofbiocompatible polymers include water-soluble polymer, such aspolyethylene glycol (PEG) or its derivatives thereof andzwitterion-containing biocompatible polymers (e.g., a phosphorylcholinecontaining polymer).

In some embodiments, the agent is an oligonucleotide, such as anti-senseoligonucleotides.

In another aspect, the invention provides a conjugate of the antibody orthe antigen binding fragment as described herein, wherein the conjugatecomprises the formula: antibody-(acyl donor glutamine-containingtag)-(linker)-(cytotoxic agent), wherein the acyl donorglutamine-containing tag is engineered at a specific site of theantibody or the antigen binding fragment (e.g., at a carboxyl terminusof the heavy or light chain, after residue T135 in the antibody heavychain, or at an another site), wherein the tag is conjugated to a linker(e.g., a linker containing one or more reactive amines (e.g., primaryamine NH₂)), and wherein the linker is conjugated to a cytotoxic agent(e.g., MMAD or other auristatins such as 0101, 0131, or 3377).

Examples of a linker containing one or more reactive amines include, butare not limited to, Ac-Lys-Gly (acetyl-lysine-glycine), aminocaproicacid, Ac-Lys-β-Ala (acetyl-lysine-β-alanine), amino-PEG2 (polyethyleneglycol)-C2, amino-PEG3-C2, amino-PEG6-C2 (or amino PEG6-propionyl),Ac-Lys-Val-Cit-PABC(acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl),amino-PEG6-C2-Val-Cit-PABC, aminocaproyl-Val-Cit-PABC,[(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC,[(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine, or Ac-Lys-putrescine.

In some embodiments, the conjugate is 1) antibody-GGLLQGPP (SEQ ID NO:474)-AcLys-VC-PABC-0101; 2) antibody-AcLys-VC-PABC-0101 and comprisesN297Q; 3) antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101 andcomprises N297Q; 4) antibody-LLQG (SEQ ID NO: 454)-amino-PEG6-C2-0131and comprises N297A; 5) antibody-LLQG (SEQ ID NO:454)-amino-PEG6-C2-3377 and comprises N297A; 6) antibody-GGLLQGA (SEQ IDNO: 475)-AcLys-VC-PABC-0101. In some embodiments, the acyl donorglutamine-containing tag comprising, e.g., GGLLQGPP (SEQ ID NO: 474) orGGLLQGA (SEQ ID NO: 475), is engineered at the C-terminus of the lightchain of the antibody. In other embodiments, the acyl donorglutamine-containing tag (e.g., LLQGA (SEQ ID NO: 463) or LLQGPP (SEQ IDNO: 472)) is engineered at the C-terminus of the heavy chain of theantibody, wherein the lysine residue at the C-terminus is deleted. Insome embodiments, the acyl donor glutamine-containing tag comprising,e.g., LLQG (SEQ ID NO: 454) is engineered after residue T135 in theantibody heavy chain or replaces amino acid residues E294-N297 in theantibody heavy chain. Examples of the antibody include, but are notlimited to, P6E01/P6E01, P6E01/H3.AQ, L1.LGF/L3.KW/P6E01;L1.LGF/L3.NY/P6E01, L1.GDF/L3.NY/P6E01, L1.LGF/L3.KW/H3.AL,L1.LGF/L3.KW/H3.AP, L1.LGF/L3.KW/H3.AQ, L1.LGF/L3.PY/H3.AP,L1.LGF/L3.PY/H3.AQ, L1.LGF/L3.NY/H3.AL, L1.LGF/L3.NY/H3.AP,L1.LGF/L3.NY/H3.AQ, L1.GDF/L3.KW/H3.AL, L1.GDF/L3.KW/H3.AP,L1.GDF/L3.KW/H3.AQ, L1.GDF/L3.PY/H3.AQ, L1.GDF/L3.NY/H3.AL,L1.GDF/L3.NY/H3.AP, L1.GDF/L3.NY/H3.AQ, L3.KW/P6E01, L3.PY/P6E01L3.NY/P6E01, L3.PY/L1.PS/P6E01, L3.PY/L1.AH/P6E01, L3.PY/L1.FF/P6E01,L3.PY/L1.PH/P6E01, L3.PY/L3.KY/P6E01, L3.PY/L3.KF/P6E01, L3.PY/H2.QR,L3.PY/H2.DY, L3.PY/H2.YQ, L3.PY/H2.LT, L3.PY/H2.HA, L3.PY/H2.QL,L3.PY/H3.YA, L3.PY/H3.AE, L3.PY/H3.AQ, L3.PY/H3.TAQ, L3.PY/P6E01,L3.PY/L1.PS/H2.QR, L3.PY/L1.PS/H2.DY, L3.PY/L1.PS/H2.YQ,L3.PY/L1.PS/H2.LT, L3.PY/L1.PS/H2.HA, L3.PY/L1.PS/H2.QL,L3.PY/L1.PS/H3.YA, L3.PY/L1.PS/H3.AE, L3.PY/L1.PS/H3.AQ,L3.PY/L1.PS/H3.TAQ, L3.PY/L1.AH/H2.QR, L3.PY/L1.AH/H2.DY,L3.PY/L1.AH/H2.YQ, L3.PY/L1.AH/H2.LT, L3.PY/L1.AH/H2.HA,L3.PY/L1.AH/H2.QL, L3.PY/L1.AH/H3.YA, L3.PY/L1.AH/H3.AE,L3.PY/L1.AH/H3.AQ, L3.PY/L1.AH/H3.TAQ, L3.PY/L1.FF/H2.QR,L3.PY/L1.FF/H2.DY, L3.PY/L1.FF/H2.YQ, L3.PY/L1.FF/H2.LT,L3.PY/L1.FF/H2.HA, L3.PY/L1.FF/H2.QL, L3.PY/L1.FF/H3.YA,L3.PY/L1.FF/H3.AE, L3.PY/L1.FF/H3.AQ, L3.PY/L1.FF/H3.TAQ,L3.PY/L1.PH/H2.QR, L3.PY/L1.PH/H2.HA, L3.PY/L1.PH/H3.AE,L3.PY/L1.PH/H3.AQ, L3.PY/L1.PH/H3.TAQ, L3.PY/L3.KY/H2.QR,L3.PY/L3.KY/H2.DY, L3.PY/L3.KY/H2.YQ L3.PY/L3.KY/H2.LT,L3.PY/L3.KY/H2.HA, L3.PY/L3.KY/H2.QL, L3.PY/L3.KY/H3.YAL3.PY/L3.KY/H3.TAQ, L3.PY/L3.KF/H2.DY, L3.PY/L3.KF/H2.YQ,L3.PY/L3.KF/H2.LT L3.PY/L3.KF/H2.QL, L3.PY/L3.KF/H3.YA,L3.PY/L3.KF/H3.AE, L3.PY/L3.KF/H3.AQ L3.PY/L3.KF/H3.TAQ, P5A2_VHVL,A02_Rd4_0.6 nM_C06, A02_Rd4_0.6 nM_C09 A02_Rd4_6 nM_C16, A02_Rd4_6nM_C03, A02_Rd4_6 nM_C01 A02_Rd4_6 nM_C26 A02_Rd4_6 nM_C25, A02_Rd4_6nM_C22, A02_Rd4_6 nM_C1 A02_Rd4_0.6 nM_C03 A02_Rd4_6 nM_C07, A02_Rd4_6nM_C23, A02_Rd4_0.6 nM_C1 A02_Rd4_6 nM_C10 A02_Rd4_6 nM_C05, A02_Rd4_0.6nM_C10, A02_Rd4_6 nM_C04, A02_Rd4_0.6 nM_C26

A02_Rd4_0.6 nM_C1 A02_Rd4_0.6 nM_C01 A02_Rd4_6 nM_C08, P5C1_VHVL,C01_Rd4_6 nM_C24, C01_Rd4_6 nM_C26, C01_Rd4_6 nM_C10, C01_Rd4_0.6 nM_C27

C01_Rd4_6 nM_C20, C01_Rd4_6 nM_C12, C01_Rd4_0.6 nM_C16, C01_Rd4_0.6nM_C09

C01_Rd4_6 nM_C09, C01_Rd4_0.6 nM_C03, C01_Rd4_0.6 nM_C06, C01_Rd4_6nM_C04

COMBO_Rd4_0.6 nM_C22, COMBO_Rd4_6 nM_C21 COMBO_Rd4_6 nM_C10,COMBO_Rd4_0.6 nM_C04, COMBO_Rd4_6 nM_C25, COMBO_Rd4_0.6 nM_C21,COMBO_Rd4_6 nM_C11, COMBO_Rd4_0.6 nM_C20, COMBO_Rd4_6 nM_C09,COMBO_Rd4_6 nM_C08, COMBO_Rd4_0.6 nM_C19, COMBO_Rd4_0.6 nM_C02,COMBO_Rd4_0.6 nM_C23, COMBO_Rd4_0.6 nM_C29, COMBO_Rd4_0.6 nM_C09,COMBO_Rd4_6 nM_C12, COMBO_Rd4_0.6 nM_C30, COMBO_Rd4_0.6 nM_C14,COMBO_Rd4_6 nM_C07, COMBO_Rd4_6 nM_C02, COMBO_Rd4_0.6 nM_C05,COMBO_Rd4_0.6 nM_C17, COMBO_Rd4_6 nM_C22, COMBO_Rd4_0.6 nM_C11,COMBO_Rd4_0.6 nM_C29, P4G4, or P1A11.

In one variation, the conjugate further comprises an amino acidsubstitution from lysine to arginine at position 222. Accordingly, forexample, the conjugate is 1) antibody-GGLLQGPP (SEQ ID NO:474)-AcLys-VC-PABC-0101 and comprises K222R; 2)antibody-AcLys-VC-PABC-0101 and comprises N297Q and K222R; 3)antibody-GGLLQGPP (SEQ ID NO: 474)-AcLys-VC-PABC-0101 and comprisesN297Q and K222R; 4) antibody-LLQG (SEQ ID NO: 454)-amino-PEG6-C2-0131and comprises N297A and K222R; 5) antibody-LLQG (SEQ ID NO:454)-amino-PEG6-C2-3377 and comprises N297A and K222R; and 6)antibody-GGLLQGA (SEQ ID NO: 475)-AcLys-VC-PABC-0101 and comprisesK222R. In some embodiments, the acyl donor glutamine-containing tagcomprising, e.g., GGLLQGPP (SEQ ID NO: 474) or GGLLQGA (SEQ ID NO: 475)is engineered at the C-terminus of the light chain of the antibody. Inother embodiments, the acyl donor glutamine-containing tag (e.g., LLQGA(SEQ ID NO: 473) or LLQGPP (SEQ ID NO: 472)) is engineered at theC-terminus of the heavy chain of the antibody, wherein the lysineresidue at the C-terminus is deleted. In some embodiments, the acyldonor glutamine-containing tag comprising, e.g., LLQG (SEQ ID NO: 454)is engineered after residue T135 in the antibody heavy chain or replacesamino acid residues E294-N297 in the antibody heavy chain. Examples ofthe antibody include, but are not limited to, P6E01/P6E01, P6E01/H3.AQ,L1.LGF/L3.KW/P6E01; L1.LGF/L3.NY/P6E01, L1.GDF/L3.NY/P6E01,L1.LGF/L3.KW/H3.AL, L1.LGF/L3.KW/H3.AP, L1.LGF/L3.KW/H3.AQ,L1.LGF/L3.PY/H3.AP, L1.LGF/L3.PY/H3.AQ, L1.LGF/L3.NY/H3.AL,L1.LGF/L3.NY/H3.AP, L1.LGF/L3.NY/H3.AQ, L1.GDF/L3.KW/H3.AL,L1.GDF/L3.KW/H3.AP, L1.GDF/L3.KW/H3.AQ, L1.GDF/L3.PY/H3.AQ,L1.GDF/L3.NY/H3.AL, L1.GDF/L3.NY/H3.AP, L1.GDF/L3.NY/H3.AQ, L3.KW/P6E01,L3.PY/P6E01, L3.NY/P6E01, L3.PY/L1.PS/P6E01, L3.PY/L1.AH/P6E01,L3.PY/L1.FF/P6E01, L3.PY/L1.PH/P6E01, L3.PY/L3.KY/P6E01,L3.PY/L3.KF/P6E01, L3.PY/H2.QR, L3.PY/H2.DY, L3.PY/H2.YQ, L3.PY/H2.LT,L3.PY/H2.HA, L3.PY/H2.QL, L3.PY/H3.YA, L3.PY/H3.AE, L3.PY/H3.AQ,L3.PY/H3.TAQ, L3.PY/P6E01, L3.PY/L1.PS/H2.QR, L3.PY/L1.PS/H2.DY,L3.PY/L1.PS/H2.YQ, L3.PY/L1.PS/H2.LT, L3.PY/L1.PS/H2.HA,L3.PY/L1.PS/H2.QL, L3.PY/L1.PS/H3.YA, L3.PY/L1.PS/H3.AE,L3.PY/L1.PS/H3.AQ, L3.PY/L1.PS/H3.TAQ, L3.PY/L1.AH/H2.QR,L3.PY/L1.AH/H2.DY, L3.PY/L1.AH/H2.YQ, L3.PY/L1.AH/H2.LT,L3.PY/L1.AH/H2.HA, L3.PY/L1.AH/H2.QL, L3.PY/L1.AH/H3.YA,L3.PY/L1.AH/H3.AE, L3.PY/L1.AH/H3.AQ, L3.PY/L1.AH/H3.TAQ,L3.PY/L1.FF/H2.QR, L3.PY/L1.FF/H2.DY, L3.PY/L1.FF/H2.YQ,L3.PY/L1.FF/H2.LT, L3.PY/L1.FF/H2.HA, L3.PY/L1.FF/H2.QL,L3.PY/L1.FF/H3.YA, L3.PY/L1.FF/H3.AE, L3.PY/L1.FF/H3.AQ,L3.PY/L1.FF/H3.TAQ, L3.PY/L1.PH/H2.QR, L3.PY/L1.PH/H2.HA,L3.PY/L1.PH/H3.AE, L3.PY/L1.PH/H3.AQ, L3.PY/L1.PH/H3.TAQ,L3.PY/L3.KY/H2.QR, L3.PY/L3.KY/H2.DY, L3.PY/L3.KY/H2.YQL3.PY/L3.KY/H2.LT, L3.PY/L3.KY/H2.HA, L3.PY/L3.KY/H2.QL,L3.PY/L3.KY/H3.YA L3.PY/L3.KY/H3.TAQ, L3.PY/L3.KF/H2.DY,L3.PY/L3.KF/H2.YQ, L3.PY/L3.KF/H2.LT L3.PY/L3.KF/H2.QL,L3.PY/L3.KF/H3.YA, L3.PY/L3.KF/H3.AE, L3.PY/L3.KF/H3.AQL3.PY/L3.KF/H3.TAQ, P5A2_VHVL, A02_Rd4_0.6 nM_C06, A02_Rd4_0.6 nM_C09A02_Rd4_6 nM_C16, A02_Rd4_6 nM_C03, A02_Rd4_6 nM_C01, A02_Rd4_6 nM_C26A02_Rd4_6 nM_C25, A02_Rd4_6 nM_C22, A02_Rd4_6 nM_C19, A02_Rd4_0.6 nM_C03A02_Rd4_6 nM_C07, A02_Rd4_6 nM_C23, A02_Rd4_0.6 nM_C18, A02_Rd4_6 nM_C10A02_Rd4_6 nM_C05, A02_Rd4_0.6 nM_C10, A02_Rd4_6 nM_C04, A02_Rd4_0.6nM_C26

A02_Rd4_0.6 nM_C13, A02_Rd4_0.6 nM_C01, A02_Rd4_6 nM_C08, P5C1_VHVL,C01_Rd4_6 nM_C24, C01_Rd4_6 nM_C26, C01_Rd4_6 nM_C10, C01_Rd4_0.6 nM_C27

C01_Rd4_6 nM_C20, C01_Rd4_6 nM_C12, C01_Rd4_0.6 nM_C16, C01_Rd4_0.6nM_C09

C01_Rd4_6 nM_C09, C01_Rd4_0.6 nM_C03, C01_Rd4_0.6 nM_C06, C01_Rd4_6nM_C04

COMBO_Rd4_0.6 nM_C22, COMBO_Rd4_6 nM_C21, COMBO_Rd4_6 nM_C10,COMBO_Rd4_0.6 nM_C04, COMBO_Rd4_6 nM_C25, COMBO_Rd4_0.6 nM_C21,COMBO_Rd4_6 nM_C11, COMBO_Rd4_0.6 nM_C20, COMBO_Rd4_6 nM_C09,COMBO_Rd4_6 nM_C08, COMBO_Rd4_0.6 nM_C19, COMBO_Rd4_0.6 nM_C02,COMBO_Rd4_0.6 nM_C23, COMBO_Rd4_0.6 nM_C29, COMBO_Rd4_0.6 nM_C09,COMBO_Rd4_6 nM_C12, COMBO_Rd4_0.6 nM_C30, COMBO_Rd4_0.6 nM_C14,COMBO_Rd4_6 nM_C07, COMBO_Rd4_6 nM_C02, COMBO_Rd4_0.6 nM_C05,COMBO_Rd4_0.6 nM_C17, COMBO_Rd4_6 nM_C22, COMBO_Rd4_0.6 nM_C11,COMBO_Rd4_0.6 nM_C29, or P4G4, or P1A11.

CD3 Antibodies and Methods of Making Thereof

The present invention further provides an antibody that binds to CD3(e.g., human CD3 (SEQ ID NO: 502; or accession number: NM_000733.3).

In one aspect, provided is an isolated antibody, or an antigen bindingfragment thereof, which specifically binds to CD3, wherein the antibodycomprises a VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown inSEQ ID NO: 320, 322, 324, 326, 328, 330, 345, 347, 349, 351, 444, 354,356, 378, 442, 380, 382, 384 386, 388, 390, 392, 394, 396, 398, or 400;and/or a light chain variable (VL) region comprising VL CDR1, VL CDR2,and VL CDR3 of the VL sequence shown in SEQ ID NO: 319, 321, 323, 325,327, 329, 344, 346, 348, 350, 352, 355, 377, 443, 445, 379, 381, 383,385, 387, 389, 391, 393, 395, 397, or 399.

In another aspect, provided is an isolated antibody, or an antigenbinding fragment thereof, which specifically binds to CD3, wherein theVH region comprises (i) a VH complementarity determining region one(CDR1) comprising the sequence shown in SEQ ID NO: 331, 332, 333, 401,402, 403, 407, 408, 415, 416, 418, 419, 420, 424, 425, 426, 446, 447, or448 (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 334, 336,337, 338, 339, 404, 405, 409, 410, 411, 412, 413, 414, 417, 418, 421,422, 427, 428, 449, or 450; and iii) a VH CDR3 comprising the sequenceshown in SEQ ID NO: 335, 406, 423, 429, or 451; and/or a light chainvariable (VL) region comprising (i) a VL CDR1 comprising the sequenceshown in SEQ ID NO: 340, 343, 430, 431, 435, or 440, 441; (ii) a VL CDR2comprising the sequence shown in SEQ ID NO: 341, 433, 452, or 436; and(iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 342, 432,434, 437, 438, 439, 446, or 453.

In some embodiments, provided is an antibody having any one of partiallight chain sequence as listed in Table 3 and/or any one of partialheavy chain sequence as listed in Table 3.

TABLE 3 mAb Light Chain Heavy Chain h2B4 DIVMTQSPDSLAVSLGERATINC TEVQLVESGGGLVQPGGSLRLSCA SSQSLFNVRSRKNYLA WYQQKP ASGFTFS DYYMTWVRQAPGKGLE GQPPKLLIS WASTRES GVPDRFS WVAFI RNRARGYT SDHNASVKGRGSGSGTDFTLTISSLQAEDVAVY FTISRDNAKNSLYLQMNSLRAEDT YC KQSYDLFT FGSGTKLEIKAVYYCAR DRPSYYVLDY WGQGTT (SEQ ID NO: 319) VTVSS (SEQ ID NO: 320) h2B4-DIVMTQSPDSLAVSLGERATINC EVQLVESGGGLVQPGGSLRLSCA VH-wt KSSQSLFNVRSRKNYLAWYQQK ASGFTFS DY YMTWVRQAPGKGLE VL_TK PGQPPKLLIS WASTRES GVPDRF WVAFIRNRARGYT SDHNASVKGR SGSGSGTDFTLTISSLQAEDVAV FTISRDNAKNSLYLQMNSLRAEDT YYCKQSYDLFT FGSGTKLEIK AVYYCAR DRPSYYVLDY WGQGTT (SEQ ID NO: 321) VTVSS(SEQ ID NO: 322) h2B4- DIVMTQSPDSLAVSLGERATINC EVQLVESGGGLVQPGGSLRLSCAVH- KSSQSLFNVRSRKNYLA WYQQK ASGFTFS DY YMTWVRQAPGKGLE hnps PGQPPKLLISWASTRES GVPDRF WVAFI RNRARGYT SDHNPSVKGR VL_TK SGSGSGTDFTLTISSLQAEDVAVFTISRDNAKNSLYLQMNSLRAEDT YYC KQSYDLFT FGSGTKLEIK AVYYCAR DRPSYYVLDYWGQGTT (SEQ ID NO: 323) VTVSS (SEQ ID NO: 324) h2B4-DIVMTQSPDSLAVSLGERATINC EVQLVESGGGLVQPGGSLRLSCA VH- KSSQSLFNVRSRKNYLAWYQQK ASGFTFS DY YMTWVRQAPGKGLE yaes PGQPPKLLIS WASTRES GVPDRF WVAFIRNRARGYT SDYAESVKGR VL_TK SGSGSGTDFTLTISSLQAEDVAVFTISRDNAKNSLYLQMNSLRAEDT YYC KQSYDLFT FGSGTKLEIK AVYYCAR DRPSYYVLDYWGQGTT (SEQ ID NO: 325) VTVSS (SEQ ID NO: 326) h2B4-DIVMTQSPDSLAVSLGERATINC EVQLVESGGGLVQPGGSLRLSCA VH- KSSQSLFNVRSRKNYLAWYQQK ASGFTFS DY YMTWVRQAPGKGLE yads PGQPPKLLIS WASTRES GVPDRF WVAFIRNRARGYT SDYADSVKGR VL_TK SGSGSGTDFTLTISSLQAEDVAVFTISRDNAKNSLYLQMNSLRAEDT YYC KQSYDLFT FGSGTKLEIK AVYYCAR DRPSYYVLDYWGQGTT (SEQ ID NO: 327) VTVSS (SEQ ID NO: 328) h2B4-DIVMTQSPDSLAVSLGERATINC T EVQLVESGGGLVQPGGSLRLSCA VH- SSQSLFNVRSRKNYLAWYQQKP ASGFTFS DY YMTWVRQAPGKGLE yaps GQPPKLLIS WASTRES GVPDRFS WVAFIRNRARGYT SDYAPSVKGR VL_TK GSGSGTDFTLTISSLQAEDVAVYFTISRDNAKNSLYLQMNSLRAEDT YC KQSYDLFT FGSGTKLEIK AVYYCAR DRPSYYVLDYWGQGTT (SEQ ID NO: 329) VTVSS (SEQ ID NO: 330) h2B4-DIVMTQSPDSLAVSLGERATINC EVQLVESGGGLVQPGGSLRLSCA VH- KSSQSLFNVRSRKNYLAWYQQK ASGFTFS DY YMTWVRQAPGKGLE hnps PGQPPKLLIY WASTRES GVPDRF WVAFIRNRARGYT SDHNPSVKGR VL_TK- SGSGSGTDFTLTISSLQAEDVAVFTISRDNAKNSLYLQMNSLRAEDT S55Y YYC KQSYDLFT FGSGTKLEIK AVYYCAR DRPSYYVLDYWGQGTT (SEQ ID NO: 344) VTVSS (SEQ ID NO: 345) h2B4-DIVMTQSPDSLAVSLGERATINC EVQLVESGGGLVQPGGSLRLSCA VH- KSSQSLFNVRSRKNYLAWYQQK ASGFTFS DY YMTWVRQAPGKGLE hnps PGQPPKLLIS WASTRES GVPDRF WVAFIRNRARGYT SDHNPSVKGR VL_TK- SGSGSGTDFTLTISSLQAEDVAVFTISRDNAKNSLYLQMNSLRAEDT S105Q YYC KQSYDLFT FGQGTKLEIK AVYYCARDRPSYYVLDY WGQGTT (SEQ ID NO: 346) VTVSS (SEQ ID NO: 347) h2B4-DIVMTQSPDSLAVSLGERATINC EVQLVESGGGLVQPGGSLRLSCA VH- KSSQSLFNVRSRKNYLAWYQQK ASGFTFS DY YMTWVRQAPGKGLE hnps PGQPPKLLIY WASTRES GVPDRF WVAFIRNRARGYT SDHNPSVKGR VL_TK- SGSGSGTDFTLTISSLQAEDVAVFTISRDNAKNSLYLQMNSLRAEDT S55Y/ YYC KQSYDLFT FGQGTKLEIK AVYYCARDRPSYYVLDY WGQGTT S105Q (SEQ ID NO: 348) VTVSS (SEQ ID NO: 349) 2B4DIVMSQSPPSLAVSVGDKVTMSC EVKLVESGGGLVQPGGSLRLSCA TSSQSLFNSRSRKNYLA WYQQKTFGFTFT D YYMTWVRQPPGKALE SGQSPKLLIS WASTRES GVPDRF WVAFI RNRARGYTSDHNASVKGR TGSGSGTDFTLTISSVQAEDLAV FTISRDNSQNILYLQMNTLRAEDS YYC KQSYDLFTFGSGTKLEIK ATYYCAR DRPSYYVLDY WGQGTT (SEQ ID NO: 350) VTVSS (SEQ ID NO:351) h2B4- DIVMTQSPDSLAVSLGERATINC T EVQLVESGGGLVQPGGSLRLSCA 11SSQSLFNSRSRKNYLA WYQQKP ASGFTFS DY YMTWVRQAPGKGLE GQPPKLLIS WASTRESGVPDRFS WVAFI RNRARGYT SDHNASVKGR GSGSGTDFTLTISSLQAEDVAVYFTISRDNAKNSLYLQMNSLRAEDT YC QQSYDTFT FGSGTKLEIK AVYYCAR DRPSYYVLDYWGQGTT (SEQ ID NO: 445) VTVSS (SEQ ID NO: 444) 1C10DIVMSQSPSSLAVSAGEKVTMSC QVQLQQPGSELVRPGASVILSCKA KSSQSLLNSRTRKNY LAWYQQKSGYTFT SY WMHWVRQRPGQGLE PGQSPKLLIY WASTRES GVPDRF WIGNI YSGGDTINYDEKFKNKAILT TGSGSGTDFTLTIDSVQPEDLAV VDTSSSTAYMHLSSLTSEDSAVYY YYCTQSFILRT FGGGTKLEIK CTR DATSRYFFDY WGQGTTVTVS (SEQ ID NO: 352) S (SEQ IDNO: 354) 1A4 DIVMSQSPSSLAVSAGEKVTMSC QVQLQQSGPDLVKPGASVEISCKKSSQSLLNSRTRKNY LAWYQQK ASGYSFT TYY LHWVRQRPGQGLE PGQSPKLLIY WASTRASGVPDRF WIGWI FPGSDN TKYNEKFKGKATL TGSGSGTDFTLTISSVQAEDLAIYTADTSSSTAYMQLSSLTSEDSAVY Y CKQSFILRT FGGGTKLEIK (SEQ FCAR NRDYYFDYWGQGTTVTVSS ID NO: 355) (SEQ ID NO: 356) 7A3 DIVVSQSPSSLAVSAGEKVIMSCEVQLQQSGAELVRPGALVKLSCK KSSQSLLNSRTRK NYLAWYQLK GSGFNIK DYYIHWVKQRPEQGLE PGQSPKLLIY SASTRES GVPDRF WIGWI DPENGN NKYDPKFQGKASITGSGSGTDFTLTISSVQTEDLAV TADTSSNIAYLQLSSLTSEDTAVYY YYC MQSFTLRTFGGGTKLEIK CAR NDNYAFDY WGQGTTVTVSS (SEQ ID NO: 443) (SEQ ID NO: 442 )25A8 QAWTQESALTTSPGEAVTLTC R EVQLVESGGGLVRPEGSLRLSCA SSTGAVTTSNYANWVQEKPDHL ASGFTFN TY AMNWVRQAPGKGLE FTGLIG GTNTRAP GVPARFSGSLI WVGRIRSKINNYA TYYAESVKGRF GDKAALTITGAQTEDEAIYFC VL TLSRDDSLSMVYLQMNSLKNEDTWYNNYWV FGGGTKLTVL (SEQ AMYYCVR HETLRSGISWFAS WGQ ID NO: 377) GTLVTVSS(SEQ ID NO: 378) 16G7 QAWTQESALTTSPGETVTLTC R EVQLVDSGGGLVQPKGSLKLSCASSTGAVTTSNYAN WVQEKPDHL ASGFTFN TY AMNWVRQAPGKGLE FTGLIG GTNNRAPGVPARFSGSL WVARI RSKSNNYA TYYADSVKDR IGDKAALTITGAQTEDEAIYFC ALFTISRDDSQSRLYLQMNNLKTEDT WYSNHWV FGGGTKLTVL (SEQ AMYYCVR HETLRSGISWFANWG ID NO: 379) QGTLVTVSS (SEQ ID NO: 380) h25A8- QAWTQEPSLTVSPGGTVTLTC REVQLVESGGGLVKPGGSLRLSCA B5 SSTGAVTTSNYAN WVQQKPGQA ASGFTFS TYAMNWVRQAPGKGLE PRGLI GGTNTRAP GTPARFSGSL WVGRI RSKINNYA TYYAESVKGRFLGGKAALTLSGAQPEDEAEYYC V TISRDDSKNTLYLQMNSLKTEDTA LWYNNYWV FGGGTKLTVL(SEQ VYYCVR HETLRSGISWFAS WGQG ID NO: 381) TLVTVSS (SEQ ID NO: 382)h25A8- QAWTQEPSLTVSPGGTVTLTC R EVQLVESGGGLVKPGGSLRLSCA B8 SSTGAVTTSNYANWVQQKPGQA ASGFTFS TY AMNWVRQAPGKGLE PRGLIG GTNTRAP GTPARFSGSL WVGRIRSKINNYA TYYAESVKGRF LGGKAALTLSGAQPEDEAEYYC V TISRDDSKNTLYLQMNSLKTEDTALWYNNHWV FGGGTKLTVL (SEQ VYYCVR HETLRSGISWFAS WGQG ID NO: 383) TLVTVSS(SEQ ID NO: 384) h25A8- QAWTQEPSLTVSPGGTVTLTC R EVQLVESGGGLVKPGGSLRLSCAB12 ASTGAVTTSNYAN WVQQKPGQ ASGFTFS TY AMNWVRQAPGKGLE APRGLIG GTNTRAPGTPARFSGS WVGRI RSKINNYA TYYAESVKGRF LLGGKAALTLSGAQPEDEAEYYCTISRDDSKNTLYLQMNSLKTEDTA VLWYNNHWV FGGGTKLTVL VYYCVR HETLRSGISWFAS WGQG(SEQ ID NO: 385) TLVTVSS (SEQ ID NO: 386) h25A8- QAWTQEPSLTVSPGGTVTLTC REVQLVESGGGLVKPGGSLRLSCA B13 TSTGAVTTSNYAN WVQQKPGQA ASGFTFS TYAMNWVRQAPGKGLE PRGLIGGTNTRAPGTPARFSGSL WVGRI RSKINNYA TYYAESVKGRFLGGKAALTLSGAQPEDEAEYYC V TISRDDSKNTLYLQMNSLKTEDTA LWYNNHWV FGGGTKLTVL(SEQ VYYCVR HETLRSGISWFAS WGQG ID NO: 387) TLVTVSS (SEQ ID NO: 388)h25A8- QAWTQEPSLTVSPGGTVTLTC R EVQLVESGGGLVKPGGSLRLSCA C5 SSTGAVTTSNYANWVQQKPGQA ASGFTFN TY AMNWVRQAPGKGLE PRGLIG GTNTRAP GTPARFSGSL WVGRIRSKINNYA TYYAESVKGRF LGGKAALTLSGAQPEDEAEYYC V TISRDDSKNTLYLQMNSLKTEDTALWYNNYWV FGGGTKLTVL (SEQ VYYCVR HETLRSGISWFAS WGQG ID NO: 389) TLVTVSS(SEQ ID NO: 390) h25A8- QAWTQEPSLTVSPGGTVTLTC R EVQLVESGGGLVKPGGSLRLSCAC8 SSTGAVTTSNYAN WVQQKPGQA ASGFTFN TY AMNWVRQAPGKGLE PRGLIG GTNTRAPGTPARFSGSL WVGRI RSKINNYA TYYAESVKGRF LGGKAALTLSGAQPEDEAEYYC VTISRDDSKNTLYLQMNSLKTEDTA LWYNNHWV FGGGTKLTVL (SEQ VYYCVR HETLRSGISWFASWGQG ID NO: 391) TLVTVSS (SEQ ID NO: 392) h25A8- QAWTQEPSLTVSPGGTVTLTC REVQLVESGGGLVKPGGSLRLSCA D13 TSTGAVTTSNYAN WVQQKPGQA ASGFTFS TYAMNWVRQAPGKGLE PRGLIGGTNTRAPGTPARFSGSL WVGRI RSHINNYA TYYAESVKGRFLGGKAALTLSGAQPEDEAEYYC V TISRDDSKNTLYLQMNSLKTEDTA LWYNNHWV FGGGTKLTVL(SEQ VYYCVR HETLRSGISWFAS WGQG ID NO: 393) TLVTVSS (SEQ ID NO: 394)h25A8- QAWTQEPSLTVSPGGTVTLTC R EVQLVESGGGLVKPGGSLRLSCA E13 TSTGAVTTSNYANWVQQKPGQA ASGFTFS TY AMNWVRQAPGKGLE PRGLIGGTNTRAPGTPARFSGSL WVGRIRSKYNNYA TYYAESVKGR LGGKAALTLSGAQPEDEAEYYC V FTISRDDSKNTLYLQMNSLKTEDTLWYNNHWV FGGGTKLTVL (SEQ AVYYCVR HETLRSGISWFAS WGQ ID NO: 395) GTLVTVSS(SEQ ID NO: 396) h25A8- QAWTQEPSLTVSPGGTVTLTC R EVQLVESGGGLVKPGGSLRLSCAF13 TSTGAVTTSNYAN WVQQKPGQA ASGFTFS TY AMNWVRQAPGKGLEPRGLIGGTNTRAPGTPARFSGSL WVGRE RSKINNYA TYYAESVKGR LGGKAALTLSGAQPEDEAEYYCV FTISRDDSKNTLYLQMNSLKTEDT LWYNNHWV FGGGTKLTVL (SEQ AVYYCVRHETLRSGISWFAS WGQ ID NO: 397) GTLVTVSS (SEQ ID NO: 398) h25A8-QAWTQEPSLTVSPGGTVTLTC R EVQLVESGGGLVKPGGSLRLSCA G13 TSTGAVTTSNYANWVQQKPGQA ASGFTFS TY AMNWVRQAPGKGLE PRGLIGGTNTRAPGTPARFSGSL WVGRIRSKINNYK TYYAESVKGRF LGGKAALTLSGAQPEDEAEYYC V TISRDDSKNTLYLQMNSLKTEDTALWYNNHWV FGGGTKLTVL (SEQ VYYCVR HETLRSGISWFAS WGQG ID NO: 399) TLVTVSS(SEQ ID NO: 400)In Table 3, the underlined sequences are CDR sequences according toKabat and in bold according to Chothia.

The invention also provides CDR portions of antibodies to CD3 (includingChothia, Kabat CDRs, and CDR contact regions). Determination of CDRregions is well within the skill of the art. It is understood that insome embodiments, CDRs can be a combination of the Kabat and Chothia CDR(also termed “combined CRs” or “extended CDRs”). In some embodiments,the CDRs are the Kabat CDRs. In other embodiments, the CDRs are theChothia CDRs. In other words, in embodiments with more than one CDR, theCDRs may be any of Kabat, Chothia, combination CDRs, or combinationsthereof. Table 4 provides examples of CDR sequences provided herein.

TABLE 4 mAb CDRH1 CDRH2 CDRH3 Heavy Chain h2B4 DYYMT (SEQ ID NO:RNRARGYT DRPSYYVLDY 331) (Kabat); (SEQ ID NO: 417) (Chothia) (SEQ ID NO:335) GFTFSDY (SEQ ID FIRNRARGYTSDHNASVKG NO: 332)(Chothia); (SEQ ID NO:334) (Kabat) GFTFSDYYMT (SEQ ID NO: 333) (Extended) h2B4- DYYMT (SEQ IDNO: RNRARGYT DRPSYYVLDY VH-wt 331) (Kabat); (SEQ ID NO: 417) (Chothia)(SEQ ID NO: 335) VL_TK GFTFSDY (SEQ ID FIRNRARGYTSDHNASVKG NO:332)(Chothia); (SEQ ID NO: 334) (Kabat) GFTFSDYYMT (SEQ ID NO: 333)(Extended) h2B4- DYYMT (SEQ ID NO: RNRARGYT DRPSYYVLDY VH-hnps 331)(Kabat); (SEQ ID NO: 417) (Chothia) (SEQ ID NO: 335) VL_TK GFTFSDY (SEQID FIRNRARGYTSDHNPSVKG NO: 332)(Chothia); (SEQ ID NO: 336) (Kabat)GFTFSDYYMT (SEQ ID NO: 333) (Extended) h2B4- DYYMT (SEQ ID NO: RNRARGYTDRPSYYVLDY VH-yaes 331) (Kabat); (SEQ ID NO: 417) (Chothia) (SEQ ID NO:335) VL_TK GFTFSDY (SEQ ID FIRNRARGYTSDYAESVKG NO: 332)(Chothia); (SEQID NO: 337) (Kabat) GFTFSDYYMT (SEQ ID NO: 333) (Extended) h2B4- DYYMT(SEQ ID NO: RNRARGYT DRPSYYVLDY VH-yads 331) (Kabat); (SEQ ID NO: 417)(Chothia) (SEQ ID NO: 335) VL_TK GFTFSDY (SEQ ID FIRNRARGYTSDYADSVKG NO:332)(Chothia); (SEQ ID NO: 338) (Kabat) GFTFSDYYMT (SEQ ID NO: 333)(Extended) h2B4- DYYMT (SEQ ID NO: RNRARGYT DRPSYYVLDY VH-yaps 331)(Kabat); (SEQ ID NO: 417) (Chothia) (SEQ ID NO: 335) VL_TK GFTFSDY (SEQID FIRNRARGYTSDYAPSVKG NO: 332)(Chothia); (SEQ ID NO: 339) (Kabat)GFTFSDYYMT (SEQ ID NO: 333) (Extended) h2B4- DYYMT (SEQ ID NO: RNRARGYTDRPSYYVLDY VH-hnps 331) (Kabat); (SEQ ID NO: 417) (Chothia) (SEQ ID NO:335) VL_TK- GFTFSDY (SEQ ID FIRNRARGYTSDHNPSVKG S55Y NO: 332)(Chothia);(SEQ ID NO: 336) (Kabat) GFTFSDYYMT (SEQ ID NO: 333) (Extended) h2B4-DYYMT (SEQ ID NO: RNRARGYT DRPSYYVLDY VH-hnps 331) (Kabat); (SEQ ID NO:417) (Chothia) (SEQ ID NO: 335) VL_TK- GFTFSDY (SEQ IDFIRNRARGYTSDHNPSVKG S105Q NO: 332)(Chothia); (SEQ ID NO: 336) (Kabat)GFTFSDYYMT (SEQ ID NO: 333) (Extended) h2B4- DYYMT (SEQ ID NO: RNRARGYTDRPSYYVLDY VH-hnps 331) (Kabat); (SEQ ID NO: 417) (Chothia) (SEQ ID NO:335) VL_TK- GFTFSDY (SEQ ID FIRNRARGYTSDHNPSVKG S55Y/ NO: 332)(Chothia);(SEQ ID NO: 336) (Kabat) S105Q GFTFSDYYMT (SEQ ID NO: 333) (Extended)2B4 DYYMT (SEQ ID NO: RNRARGYT DRPSYYVLDY 331) (Kabat); (SEQ ID NO: 417)(Chothia) (SEQ ID NO: 335) GFTFTDY (SEQ ID FIRNRARGYTSDHNASVKG NO:415)(Chothia); (SEQ ID NO: 418) (Kabat) GFTFTDYYMT (SEQ ID NO: 416)(Extended) h2B4-11 DYYMT (SEQ ID NO: RNRARGYT DRPSYYVLDY 331) (Kabat);(SEQ ID NO: 417) (Chothia) (SEQ ID NO: 335) GFTFSDY (SEQ IDFIRNRARGYTSDHNASVKG NO: 332)(Chothia); (SEQ ID NO: 418) (Kabat)GFTFSDYYMT (SEQ ID NO: 333) (Extended) 1C10 SYWMH (SEQ ID NO: YSGGDTDATSRYFFDY 418) (Kabat) (SEQ ID NO: 421) (Chothia) (SEQ ID NO: 423)GYTFTSY (SEQ ID NIYSGGDTINYDEKFKN NO: 419) (Chothia) (SEQ ID NO: 422)(Kabat) GYTFTSYWMH (SEQ ID NO: 420) (Extended) 1A4 TYYLH (SEQ ID NO:FPGSDN (SEQ ID NO: 427) NRDYYFDY 424) (Kabat) (Chothia) (SEQ ID NO: 429)GYSFTTYY (SEQ ID WIFPGSDNTKYNEKFKG NO: 425) (Chothia) (SEQ ID NO: 428)(Kabat) GYSFTTYYLH (SEQ ID NO: 426) (Extended) 7A3 DYYIH (SEQ ID NO:DPENGN (SEQ ID NO: 449) NDNYAFDY 446) (Kabat) (Chothia) (SEQ ID NO: 451)GFNIKDY(SEQ ID WIDPENGNNKYDPKFQG NO: 447) (Chothia) (SEQ ID NO: 450)(Kabat) GFNIKDYYIH (SEQ ID NO: 448) (Extended) 25A8 TYAMN (SEQ ID NO:RSKINNYA (SEQ ID NO: HETLRSGISWFA 401) (Kabat); 404) (Chothia) S (SEQ IDNO: GFTFNTY (SEQ ID RIRSKINNYATYYAESVKG 406) NO: 402)(Chothia); (SEQ IDNO: 405) (Kabat) GFTFNTYAMN (SEQ ID NO: 403) (Extended) 16G7 TYAMN (SEQID NO: RSKSNNYA (SEQ ID NO: HETLRSGISWFA 401) (Kabat); 404) (Chothia) N(SEQ ID NO: GFTFNTY (SEQ ID RIRSKSNNYATYYADSVKD 406) NO: 402)(Chothia);(SEQ ID NO: 405) (Kabat) GFTFNTYAMN (SEQ ID NO: 403) (Extended) h25A8-TYAMN (SEQ ID NO: RSKINNYA (SEQ ID NO: HETLRSGISWFA B5 401) (Kabat);404) (Chothia) S (SEQ ID NO: GFTFSTY (SEQ ID RIRSKINNYATYYAESVKG 406)NO: 407)(Chothia); (SEQ ID NO: 405) (Kabat) GFTFSTYAMN (SEQ ID NO: 408)(Extended) h25A8- TYAMN (SEQ ID NO: RSKINNYA (SEQ ID NO: HETLRSGISWFA B8401) (Kabat); 404) (Chothia) S (SEQ ID NO: GFTFSTY (SEQ IDRIRSKINNYATYYAESVKG 406) NO: 407)(Chothia); (SEQ ID NO: 405) (Kabat)GFTFSTYAMN (SEQ ID NO: 408) (Extended) h25A8- TYAMN (SEQ ID NO: RSKINNYA(SEQ ID NO: HETLRSGISWFA B12 401) (Kabat); 404) (Chothia) S (SEQ ID NO:GFTFSTY (SEQ ID RIRSKINNYATYYAESVKG 406) NO: 407)(Chothia); (SEQ ID NO:405) (Kabat) GFTFSTYAMN (SEQ ID NO: 408) (Extended) h25A8- TYAMN (SEQ IDNO: RSKINNYA (SEQ ID NO: HETLRSGISWFA B13 401) (Kabat); 404) (Chothia) S(SEQ ID NO: GFTFSTY (SEQ ID RIRSKINNYATYYAESVKG 406) NO: 407)(Chothia);(SEQ ID NO: 405) (Kabat) GFTFSTYAMN (SEQ ID NO: 408) (Extended) h25A8-TYAMN (SEQ ID NO: RSKINNYA (SEQ ID NO: HETLRSGISWFA C5 401) (Kabat);404) (Chothia) S (SEQ ID NO: GFTFNTY (SEQ ID RIRSKINNYATYYAESVKG 406)NO: 402)(Chothia); (SEQ ID NO: 405) (Kabat) GFTFNTYAMN (SEQ ID NO: 403)(Extended) h25A8- TYAMN (SEQ ID NO: RSKINNYA (SEQ ID NO: HETLRSGISWFA C8401) (Kabat); 404) (Chothia) S (SEQ ID NO: GFTFNTY (SEQ IDRIRSKINNYATYYAESVKG 406) NO: 402)(Chothia); (SEQ ID NO: 405) (Kabat)GFTFNTYAMN (SEQ ID NO: 403) h25A8- TYAMN (SEQ ID NO: RSHINNYA (SEQ IDNO: HETLRSGISWFA D13 401) (Kabat); 409) (Chothia) S (SEQ ID NO: GFTFSTY(SEQ ID RIRSHINNYATYYAESVKG 406) NO: 407)(Chothia); (SEQ ID NO: 410)(Kabat) GFTFSTYAMN (SEQ ID NO: 408) (Extended) h25A8- TYAMN (SEQ ID NO:RSKYNNYA (SEQ ID NO: HETLRSGISWFA E13 401) (Kabat); 411) (Chothia) S(SEQ ID NO: GFTFSTY (SEQ ID RIRSKYNNYATYYAESVKG 406) NO: 407)(Chothia);(SEQ ID NO: 412) (Kabat) GFTFSTYAMN (SEQ ID NO: 408) (Extended) h25A8-TYAMN (SEQ ID NO: RSKINNYA (SEQ ID NO: HETLRSGISWFA F13 401) (Kabat);404) (Chothia) S (SEQ ID NO: GFTFSTY (SEQ ID RERSKINNYATYYAESVKG 406)NO: 407)(Chothia); (SEQ ID NO: 413) (Kabat) GFTFSTYAMN (SEQ ID NO: 408)(Extended) h25A8- TYAMN (SEQ ID NO: RSKINNYA (SEQ ID NO: HETLRSGISWFAG13 401) (Kabat); 404) (Chothia) S (SEQ ID NO: GFTFSTY (SEQ IDRIRSKINNYKTYYAESVKG 406) NO: 407)(Chothia); (SEQ ID NO: 414) (Kabat)GFTFSTYAMN (SEQ ID NO: 408) (Extended) Light Chain h2B4 TSSQSLFNVRSRKNWASTRES KQSYDLFT YLA (SEQ ID NO: 341) (SEQ ID NO: 342) (SEQ ID NO: 340)h2B4- KSSQSLFNVRSRKN WASTRES KQSYDLFT VH-wt YLA (SEQ ID NO: 341) (SEQ IDNO: 342) VL_TK (SEQ ID NO: 343) h2B4- KSSQSLFNVRSRKN WASTRES KQSYDLFTVH-hnps YLA (SEQ ID NO: 341) (SEQ ID NO: 342) VL_TK (SEQ ID NO: 343)h2B4- KSSQSLFNVRSRKN WASTRES KQSYDLFT VH-yaes YLA (SEQ ID NO: 341) (SEQID NO: 342) VL_TK (SEQ ID NO: 343) h2B4- KSSQSLFNVRSRKN WASTRES KQSYDLFTVH-yads YLA (SEQ ID NO: 341) (SEQ ID NO: 342) VL_TK (SEQ ID NO: 343)h2B4- KSSQSLFNVRSRKN WASTRES KQSYDLFT VH-yaps YLA (SEQ ID NO: 341) (SEQID NO: 342) VL_TK (SEQ ID NO: 343) 2B4 TSSQSLFNSRSRKN WASTRES KQSYDLFTYLA (SEQ ID NO: (SEQ ID NO: 341) (SEQ ID NO: 342) 430) h2B4-11TSSQSLFNSRSRKN WASTRES QQSYDTFT YLA (SEQ ID NO: (SEQ ID NO: 341) (SEQ IDNO: 446) 430) 1C10 KSSQSLLNSRTRKN WASTRES TQSFILRT (SEQ Y (SEQ ID NO:431) (SEQ ID NO: 341) ID NO: 432) 1A4 KSSQSLLNSRTRKN WASTRAS KQSFILRT(SEQ Y (SEQ ID NO: 431) (SEQ ID NO: 433) ID NO: 434) 7A3 KSSQSLLNSRTRKNSASTRES MQSFTLRT Y (SEQ ID NO: 431) (SEQ ID NO: 452) (SEQ ID NO: 453)h2B4- KSSQSLFNVRSRKN WASTRES KQSYDLFT VH-hnps YLA (SEQ ID NO: 341) (SEQID NO: 342) VL_TK- (SEQ ID NO: 343) S55Y h2B4- KSSQSLFNVRSRKN WASTRESKQSYDLFT VH-hnps YLA (SEQ ID NO: 341) (SEQ ID NO: 342) VL_TK- (SEQ IDNO: 343) S105Q h2B4- KSSQSLFNVRSRKN WASTRES KQSYDLFT VH-hnps YLA (SEQ IDNO: 341) (SEQ ID NO: 342) VL_TK- (SEQ ID NO: 343) S55Y/ S105Q 25A8RSSTGAVTTSNYAN GTNTRAP (SEQ ID NO: VLWYNNYWV (SEQ ID NO: 435) 436) (SEQID NO: 437) 16G7 RSSTGAVTTSNYAN GTNTRAP (SEQ ID NO: ALWYSNHWV (SEQ IDNO: 435) 436) (SEQ ID NO: 438) h25A8- RSSTGAVTTSNYAN GTNTRAP (SEQ ID NO:VLWYNNYWV B5 (SEQ ID NO: 435) 436) (SEQ ID NO: 437) h25A8-RSSTGAVTTSNYAN GTNTRAP (SEQ ID NO: VLWYNNHWV B8 (SEQ ID NO: 435) 436)(SEQ ID NO: 439) h25A8- RASTGAVTTSNYAN GTNTRAP (SEQ ID NO: VLWYNNHWV B12(SEQ ID NO: 440) 436) (SEQ ID NO: 439) h25A8- RTSTGAVTTSNYAN GTNTRAP(SEQ ID NO: VLWYNNHWV B13 (SEQ ID NO: 441) 436) (SEQ ID NO: 439) h25A8-RSSTGAVTTSNYAN GTNTRAP (SEQ ID NO: VLWYNNYWV C5 (SEQ ID NO: 435) 436)(SEQ ID NO: 437) h25A8- RSSTGAVTTSNYAN GTNTRAP (SEQ ID NO: VLWYNNHWV C8(SEQ ID NO: 435) 436) (SEQ ID NO: 439) h25A8- RTSTGAVTTSNYAN GTNTRAP(SEQ ID NO: VLWYNNHWV D13 (SEQ ID NO: 441) 436) (SEQ ID NO: 439) h25A8-RTSTGAVTTSNYAN GTNTRAP (SEQ ID NO: VLWYNNHWV E13 (SEQ ID NO: 441) 436)(SEQ ID NO: 439) h25A8- RTSTGAVTTSNYAN GTNTRAP (SEQ ID NO: VLWYNNHWV F13(SEQ ID NO: 441) 436) (SEQ ID NO: 439) h25A8- RTSTGAVTTSNYAN GTNTRAP(SEQ ID NO: VLWYNNHWV G13 (SEQ ID NO: 441) 436) (SEQ ID NO: 439)

The invention also provides isolated polynucleotides encoding theantibodies of the invention, and vectors and host cells comprising thepolynucleotide.

In one embodiment, a polynucleotide comprises a sequence encoding theheavy chain and/or the light chain variable regions of antibody h2B4,h2B4-VH-wt VL_TK, h2B4-VH-hnps VL_TK, h2B4-VH-yaes VL_TK, h2B4-VH-yadsVL_TK, h2B4-VH-yaps VL_TK, h2B4-VH-hnps VL_TK-S55Y, h2B4-VH-hnpsVL_TK-S105Q, h2B4-vH-hnps VL_TK-S55Y/S105Q, 2B4, h2B4-11, 1C10, 1A4,7A3, 25A8, 16G7, h25A8-B5, h25A8-B8, h25A8-B12, h25A8-B13, h25A8-05,h25A8-C8, h25A8-D13, h25A8-E13, h25A8-F13, or h25A8-G13. The sequenceencoding the antibody of interest may be maintained in a vector in ahost cell and the host cell can then be expanded and frozen for futureuse. Vectors (including expression vectors) and host cells are furtherdescribed herein.

The invention also encompasses fusion proteins comprising one or morefragments or regions from the antibodies of this invention. In oneembodiment, a fusion polypeptide is provided that comprises at least 10contiguous amino acids of the variable light chain region shown in SEQID NOs: 319, 321, 323, 325, 327, 329, 344, 346, 348, 350, 445, 352, 355,443, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, or 399,and/or at least 10 amino acids of the variable heavy chain region shownin SEQ ID NOs: 320, 322, 324, 326, 328, 330, 345, 347, 349, 351, 354,356, 444, 442, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, or400. In other embodiments, a fusion polypeptide is provided thatcomprises at least about 10, at least about 15, at least about 20, atleast about 25, or at least about 30 contiguous amino acids of thevariable light chain region and/or at least about 10, at least about 15,at least about 20, at least about 25, or at least about 30 contiguousamino acids of the variable heavy chain region. In another embodiment,the fusion polypeptide comprises a light chain variable region and/or aheavy chain variable region, as shown in any of the sequence pairsselected from among SEQ ID NOs: 319 and 320, 321 and 322, 323 and 324,325 and 326, 327 and 328, 329 and 330, 344 and 345, 346 and 347, 348 and349, 350 and 351, 445 and 444, 352 and 354, 355 and 356, 443 and 442,377 and 378, 379 and 380, 381 and 382, 383 and 384, 385 and 386, 387 and388, 389 and 390, 391 and 392, 393 and 394, 395 and 396, 397 and 398, or399 and 400. In another embodiment, the fusion polypeptide comprises oneor more CDR(s). In still other embodiments, the fusion polypeptidecomprises CDR H3 (VH CDR3) and/or CDR L3 (VL CDR3). For purposes of thisinvention, a fusion protein contains one or more antibodies and anotheramino acid sequence to which it is not attached in the native molecule,for example, a heterologous sequence or a homologous sequence fromanother region. Exemplary heterologous sequences include, but are notlimited to a “tag” such as a FLAG tag or a 6His tag. Tags are well knownin the art.

A fusion polypeptide can be created by methods known in the art, forexample, synthetically or recombinantly. Typically, the fusion proteinsof this invention are made by preparing an expressing a polynucleotideencoding them using recombinant methods described herein, although theymay also be prepared by other means known in the art, including, forexample, chemical synthesis.

Representative materials of the CD3 antibody in the present inventionwere deposited in the American Type Culture Collection (ATCC) on Sep.11, 2015. Vector having ATCC Accession No. PTA-122513 is apolynucleotide encoding a humanized CD3 antibody heavy chain variableregion, and vector having ATCC Accession No. PTA-122512 is apolynucleotide encoding a humanized CD3 antibody light chain variableregion. The deposits were made under the provisions of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Pfizer, Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the culture of the deposit to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 U.S.C. Section 122 and the Commissioner's rules pursuant thereto(including 37 C.F.R. Section 1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

Bispecific Antibodies and Methods of Making

Bispecific antibodies, monoclonal antibodies that have bindingspecificities for at least two different antigens, can be prepared usingthe antibodies disclosed herein. Methods for making bispecificantibodies are known in the art (see, e.g., Suresh et al., Methods inEnzymology 121:210, 1986). Traditionally, the recombinant production ofbispecific antibodies was based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, with the two heavy chainshaving different specificities (Millstein and Cuello, Nature 305,537-539, 1983).

According to one approach to making bispecific antibodies, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantregion sequences. The fusion preferably is with an immunoglobulin heavychain constant region, comprising at least part of the hinge, CH2 andCH3 regions. It is preferred to have the first heavy chain constantregion (CH1), containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are cotransfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In one approach, the bispecific antibodies are composed of a hybridimmunoglobulin heavy chain with a first binding specificity in one arm,and a hybrid immunoglobulin heavy chain-light chain pair (providing asecond binding specificity) in the other arm. This asymmetric structure,with an immunoglobulin light chain in only one half of the bispecificmolecule, facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations. This approach isdescribed in PCT Publication No. WO 94/04690.

In another approach, the bispecific antibodies are composed of aminoacid modification in the first hinge region in one arm, and thesubstituted/replaced amino acid in the first hinge region has anopposite charge to the corresponding amino acid in the second hingeregion in another arm. This approach is described in InternationalPatent Application No. PCT/US2011/036419 (WO2011/143545).

In another approach, the formation of a desired heteromultimeric orheterodimeric protein (e.g., bispecific antibody) is enhanced byaltering or engineering an interface between a first and a secondimmunoglobulin-like Fc region (e.g., a hinge region and/or a CH3region). In this approach, the bispecific antibodies may be composed ofa CH3 region, wherein the CH3 region comprises a first CH3 polypeptideand a second CH3 polypeptide which interact together to form a CH3interface, wherein one or more amino acids within the CH3 interfacedestabilize homodimer formation and are not electrostaticallyunfavorable to homodimer formation. This approach is described inInternational Patent Application No. PCT/US2011/036419 (WO2011/143545).

In another approach, the bispecific antibodies can be generated using aglutamine-containing peptide tag engineered to the antibody directed toan epitope (e.g., BCMA) in one arm and another peptide tag (e.g., aLys-containing peptide tag or a reactive endogenous Lys) engineered to asecond antibody directed to a second epitope in another arm in thepresence of transglutaminase. This approach is described inInternational Patent Application No. PCT/IB2011/054899 (WO2012/059882).

In another aspect of the invention, the heterodimeric protein (e.g.,bispecific antibody) as described herein comprises a full-length humanantibody, wherein a first antibody variable domain of the heterodimericprotein is capable of recruiting the activity of a human immune effectorcell by specifically binding to an effector antigen located on the humanimmune effector cell, and wherein a second antibody variable domain ofthe heterodimeric protein is capable of specifically binding to a targetantigen. In some embodiments, the human antibody has an IgG1, IgG2,IgG3, or IgG4 isotype. In some embodiments, the heterodimeric proteincomprises an immunologically inert Fc region.

The human immune effector cell can be any of a variety of immuneeffector cells known in the art. For example, the immune effector cellcan be a member of the human lymphoid cell lineage, including, but notlimited to, a T cell (e.g., a cytotoxic T cell), a B cell, and a naturalkiller (NK) cell. The immune effector cell can also be, for examplewithout limitation, a member of the human myeloid lineage, including,but not limited to, a monocyte, a neutrophilic granulocyte, and adendritic cell. Such immune effector cells may have either a cytotoxicor an apoptotic effect on a target cell or other desired effect uponactivation by binding of an effector antigen.

The effector antigen is an antigen (e.g., a protein or a polypeptide)that is expressed on the human immune effector cell. Examples ofeffector antigens that can be bound by the heterodimeric protein (e.g.,a heterodimeric antibody or a bispecific antibody) include, but are notlimited to, human CD3 (or CD3 (Cluster of Differentiation) complex),CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD89.

The target cell can be a cell that is native or foreign to humans. In anative target cell, the cell may have been transformed to be a malignantcell or pathologically modified (e.g., a native target cell infectedwith a virus, a plasmodium, or a bacterium). In a foreign target cell,the cell is an invading pathogen, such as a bacterium, a plasmodium, ora virus.

The target antigen is expressed on a target cell in a diseased condition(e.g., an inflammatory disease, a proliferative disease (e.g., cancer),an immunological disorder, a neurological disease, a neurodegenerativedisease, an autoimmune disease, an infectious disease (e.g., a viralinfection or a parasitic infection), an allergic reaction, agraft-versus-host disease or a host-versus-graft disease). A targetantigen is not effector antigen. Examples of the target antigensinclude, but are not limited to, BCMA, EpCAM (Epithelial Cell AdhesionMolecule), CCR5 (Chemokine Receptor type 5), CD19, HER (Human EpidermalGrowth Factor Receptor)-2/neu, HER-3, HER-4, EGFR (Epidermal GrowthFactor Receptor), PSMA, CEA, MUC-1 (Mucin), MUC2, MUC3, MUC4, MUC5AC,MUC5B, MUC7, ClhCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3,9-O-Acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA, GD2, CarboanhydraseIX (MN/CA IX), CD44v6, Shh (Sonic Hedgehog), Wue-1, Plasma Cell Antigen,(membrane-bound) IgE, MCSP (Melanoma Chondroitin Sulfate Proteoglycan),CCR8, TNF-alpha precursor, STEAP, mesothelin, A33 Antigen, PSCA(Prostate Stem Cell Antigen), Ly-6; desmoglein 4, E-cadherin neoepitope,Fetal Acetylcholine Receptor, CD25, CA19-9 marker, CA-125 marker and MIS(Muellerian Inhibitory Substance) Receptor type II, sTn (sialylated Tnantigen; TAG-72), FAP (fibroblast activation antigen), endosialin,EGFRvIII, LG, SAS and CD63.

In some embodiments, the heterodimeric protein (e.g., bispecificantibody) as described herein comprises a full-length human antibody,wherein a first antibody variable domain of the heterodimeric protein iscapable of recruiting the activity of a human immune effector cell byspecifically binding to an effector antigen (e.g., CD3 antigen) locatedon the human immune effector cell, wherein a second antibody variabledomain of the heterodimeric protein is capable of specifically bindingto a target antigen (e.g., CD20 antigen or EpCAM), wherein the first andsecond antibody variable domain of the heterodimeric protein compriseamino acid modifications at positions 223, 225, and 228 (e.g., (C223E orC223R), (E225R), and (P228E or P228R)) in the hinge region and atposition 409 or 368 (e.g., K409R or L368E (EU numbering scheme)) in theCH3 region of human IgG2 (SEQ ID NO: 493).

In some embodiments, the first and second antibody variable domains ofthe heterodimeric protein comprise amino acid modifications at positions221 and 228 (e.g., (D221R or D221E) and (P228R or P228E)) in the hingeregion and at position 409 or 368 (e.g., K409R or L368E (EU numberingscheme)) in the CH3 region of human IgG1 (SEQ ID NO: 494).

In some embodiments, the first and second antibody variable domains ofthe heterodimeric protein comprise amino acid modifications at positions228 (e.g., (P228E or P228R)) in the hinge region and at position 409 or368 (e.g., R409 or L368E (EU numbering scheme)) in the CH3 region ofhuman IgG4 (SEQ ID NO: 495).

In another embodiment, the first antibody variable domain of theheterodimeric protein comprises a VH region comprising a VH CDR1, VHCDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 320, 322, 324,326, 328, 330, 345, 347, 349, 351, 444, 354, 356, 378, 442, 380, 382,384 386, 388, 390, 392, 394, 396, 398, or 400; and/or a light chainvariable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 of the VLsequence shown in SEQ ID NO: 319, 321, 323, 325, 327, 329, 344, 346,348, 350, 352, 355, 377, 443, 445, 379, 381, 383, 385, 387, 389, 391,393, 395, 397, or 399, and the second antibody variable domain of theheterodimeric protein comprises VH region comprising the VH sequenceshown in SEQ ID NO: 2, 3, 7, 8, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,35, 37, 39, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 83, 87, 92, 95, 97, 99, 101, 104, 106, 110, 112, 114,118, 120, 122, 125, 127, 313, 314, 363, or 365; and/or a VL regioncomprising VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQID NO: 1, 4, 5, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 317, 81, 82, 84, 85, 86, 88, 89, 90, 91,93, 94, 96, 98, 100, 102, 103, 105, 107, 108, 109, 111, 113, 115, 116,117, 119, 121, 123, 124, 126, 128, 315, or 364.

In another embodiment, the first antibody variable domain comprises aheavy chain variable (VH) region comprising a VH CDR1, VH CDR2, and VHCDR3 of the VH sequence shown in SEQ ID NO: 324 or 388; and/or a lightchain variable (VL) region comprising VL CDR1, VL CDR2, and VL CDR3 ofthe VL sequence shown in SEQ ID NO: 323 or 387; and the second antibodyvariable domain comprises a heavy chain variable (VH) region comprisinga VH CDR1, VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO:112; and/or a light chain variable (VL) region comprising VL CDR1, VLCDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 38.

In another embodiment, the first antibody variable domain comprises aheavy chain variable (VH) region comprising (i) a VH complementaritydetermining region one (CDR1) comprising the sequence shown in SEQ IDNO: 331 (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 417;and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 335; anda light chain variable (VL) region comprising (i) a VL CDR1 comprisingthe sequence shown in SEQ ID NO: 343; (ii) a VL CDR2 comprising thesequence shown in SEQ ID NO: 341; and (iii) a VL CDR3 comprising thesequence shown in SEQ ID NO: 342; and the second antibody variabledomain comprises a heavy chain VH region comprising a heavy chainvariable (VH) region comprising (i) a VH CDR1 comprising the sequenceshown in SEQ ID NO: 156; (ii) a VH CDR2 comprising the sequence shown inSEQ ID NO: 159; and (iii) a VH CDR3 comprising SEQ ID NO: 155; and alight chain variable (VL) region comprising (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 209; (ii) a VL CDR2 comprising the sequenceshown in SEQ ID NO: 221; and (iii) a VL CDR3 comprising the sequenceshown in SEQ ID NO: 225. The VH CDR1 of the first antibody variabledomain may further comprise the sequence of S, FS, TFS, FTFS or GFTFSimmediately preceding the sequence shown in SEQ ID NO: 331. For example,the VH CDR1 of the first antibody variable domain may comprise thesequence shown in SEQ ID NO: 333. The VH CDR2 of the first antibodyvariable domain may further comprise the sequence of F or FI immediatelypreceding the sequence shown in SEQ ID NO: 417 and/or the sequence of S,SD, SDH, SDHN, SDHNP, SDHNPS, SDHNPSV, SDHNPSVK, or SDHNPSVKGimmediately following the sequence shown in SEQ ID NO: 417. For example,the VH CDR2 of the first antibody variable domain may comprise thesequence shown in SEQ ID NO: 336. The VH CDR1 of the second antibodyvariable domain may further comprise the sequence of S, FS, TFS, FTFS orGFTFS immediately preceding the sequence shown in SEQ ID NO: 156. Forexample, the VH CDR1 of the second antibody variable domain may comprisethe sequence shown in SEQ ID NO: 157. The VH CDR2 of the second antibodyvariable domain may further comprise the sequence of A or AI immediatelypreceding the sequence shown in SEQ ID NO: 159 and/or the sequence of L,LP, LPY, LPYA, LPYAD, LPYADS, LPYADSV, LPYADSVK, or LPYADSVKGimmediately following the sequence shown in SEQ ID NO: 159. For example,the VH CDR2 of the second antibody variable domain may comprise thesequence shown in SEQ ID NO: 158.

In another embodiment, the first antibody variable domain comprises aheavy chain variable (VH) region comprising (i) a VH complementaritydetermining region one (CDR1) comprising the sequence shown in SEQ IDNO: 332 (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 336;and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 335; anda light chain variable (VL) region comprising (i) a VL CDR1 comprisingthe sequence shown in SEQ ID NO: 343; (ii) a VL CDR2 comprising thesequence shown in SEQ ID NO: 341; and (iii) a VL CDR3 comprising thesequence shown in SEQ ID NO: 342; and the second antibody variabledomain comprises a heavy chain VH region comprising a heavy chainvariable (VH) region comprising (i) a VH CDR1 comprising the sequenceshown in SEQ ID NO: 151; (ii) a VH CDR2 comprising the sequence shown inSEQ ID NO: 158; and (iii) a VH CDR3 comprising SEQ ID NO: 155; and alight chain variable (VL) region comprising (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 209; (ii) a VL CDR2 comprising the sequenceshown in SEQ ID NO: 221; and (iii) a VL CDR3 comprising the sequenceshown in SEQ ID NO: 225. The VH CDR1 of the first antibody variabledomain may further comprise the sequence of Y, YM or YMT immediatelyfollowing the sequence shown in SEQ ID NO: 332. For example, the VH CDR1of the first antibody variable domain may comprise the sequence shown inSEQ ID NO: 333. The VH CDR1 of the second antibody variable domain mayfurther comprise the sequence P, PM, or PMS immediately following thesequence shown in SEQ ID NO: 151. For example, the VH CDR1 of the secondantibody variable domain may comprise the sequence shown in SEQ ID NO:157.

In another embodiment, the first antibody variable domain comprises aheavy chain variable (VH) region comprising (i) a VH complementaritydetermining region one (CDR1) comprising the sequence shown in SEQ IDNO: 401 (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 404;and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 406; anda light chain variable (VL) region comprising (i) a VL CDR1 comprisingthe sequence shown in SEQ ID NO: 441; (ii) a VL CDR2 comprising thesequence shown in SEQ ID NO: 436; and (iii) a VL CDR3 comprising thesequence shown in SEQ ID NO: 439; and the second antibody variabledomain comprises a heavy chain VH region comprising a heavy chainvariable (VH) region comprising (i) a VH CDR1 comprising the sequenceshown in SEQ ID NO: 156; (ii) a VH CDR2 comprising the sequence shown inSEQ ID NO: 159; and (iii) a VH CDR3 comprising SEQ ID NO: 155; and alight chain variable (VL) region comprising (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 209; (ii) a VL CDR2 comprising the sequenceshown in SEQ ID NO: 221; and (iii) a VL CDR3 comprising the sequenceshown in SEQ ID NO: 225. The VH CDR1 of the first antibody variabledomain may further comprise the sequence of S, FS, TFS, FTFS or GFTFSimmediately preceding the sequence shown in SEQ ID NO: 401. For example,the VH CDR1 of the first antibody variable domain comprises the sequenceshown in SEQ ID NO: 408. The VH CDR2 of the first antibody variabledomain may further comprise the sequence of R or RI immediatelypreceding the sequence shown in SEQ ID NO: 404 and/or the sequence of T,TY, TYY, TYYA, TYYAE, TYYAES, TYYAESV, TYYAESVK, or TYYAESVKGimmediately following the sequence shown in SEQ ID NO: 404. For example,the bispecific antibody of claim 49, wherein the VH CDR2 of the fristantibody variable domain comprises the sequence shown in SEQ ID NO: 405.The VH CDR1 of the second antibody variable domain may further comprisethe sequence of S, FS, TFS, FTFS or GFTFS immediately preceding thesequence shown in SEQ ID NO: 156. For example, the VH CDR1 of the secondantibody variable domain comprises the sequence shown in SEQ ID NO: 157.The VH CDR2 of the second antibody variable domain may further comprisethe sequence of A or AI immediately preceding the sequence shown in SEQID NO: 159 and/or the sequence of L, LP, LPY, LPYA, LPYAD, LPYADS,LPYADSV, LPYADSVK, or LPYADSVKG immediately following the sequence shownin SEQ ID NO: 159. For example, the VH CDR2 of the second antibodyvariable domain may comprise the sequence shown in SEQ ID NO: 158.

In another embodiment, the first antibody variable domain comprises aheavy chain variable (VH) region comprising (i) a VH complementaritydetermining region one (CDR1) comprising the sequence shown in SEQ IDNO: 407 (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 405;and iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: 406; anda light chain variable (VL) region comprising (i) a VL CDR1 comprisingthe sequence shown in SEQ ID NO: 441; (ii) a VL CDR2 comprising thesequence shown in SEQ ID NO: 436; and (iii) a VL CDR3 comprising thesequence shown in SEQ ID NO: 439; and the second antibody variabledomain comprises a heavy chain VH region comprising a heavy chainvariable (VH) region comprising (i) a VH CDR1 comprising the sequenceshown in SEQ ID NO: 151; (ii) a VH CDR2 comprising the sequence shown inSEQ ID NO: 158; and (iii) a VH CDR3 comprising SEQ ID NO: 155; and alight chain variable (VL) region comprising (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 209; (ii) a VL CDR2 comprising the sequenceshown in SEQ ID NO: 221; and (iii) a VL CDR3 comprising the sequenceshown in SEQ ID NO: 225. The VH CDR1 of the first antibody variabledomain may further comprise the sequence of A, AM or AMN immediatelyfollowing the sequence shown in SEQ ID NO: 407. For example, the VH CDR1of the first antibody variable domain may comprise the sequence shown inSEQ ID NO: 408. The VH CDR1 of the second antibody variable domain mayfurther comprise the sequence P, PM, or PMS immediately following thesequence shown in SEQ ID NO: 151. For example, the VH CDR1 of the secondantibody variable domain may comprise the sequence shown in SEQ ID NO:157.

The antibodies useful in the present invention can encompass monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab′,F(ab′)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies,heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion (e.g., a domain antibody),humanized antibodies, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site ofthe required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. The antibodies may be murine, rat, human, or anyother origin (including chimeric or humanized antibodies).

In some embodiments, the BCMA or CD3 antibody as described herein is amonoclonal antibody. For example, the BCMA or CD3 antibody is ahumanized monoclonal antibody or a chimeric monoclonal antibody.

In some embodiments, the antibody comprises a modified constant region,such as, for example without limitation, a constant region that hasincreased potential for provoking an immune response. For example, theconstant region may be modified to have increased affinity to an Fcgamma receptor such as, e.g., FcγRI, FcγRIIA, or FcγIII.

In some embodiments, the antibody comprises a modified constant region,such as a constant region that is immunologically inert, that is, havinga reduced potential for provoking an immune response. In someembodiments, the constant region is modified as described in Eur. J.Immunol., 29:2613-2624, 1999; PCT Application No. PCT/GB99/01441; and/orUK Patent Application No. 98099518. The Fc can be human IgG1, humanIgG2, human IgG3, or human IgG4. The Fc can be human IgG2 containing themutation A330P331 to S330S331 (IgG2Δa), in which the amino acid residuesare numbered with reference to the wild type IgG2 sequence. Eur. J.Immunol., 29:2613-2624, 1999. In some embodiments, the antibodycomprises a constant region of IgG₄ comprising the following mutations(Armour et al., Molecular Immunology 40 585-593, 2003): E233F234L235 toP233V234A235 (IgG4Δc), in which the numbering is with reference to wildtype IgG4. In yet another embodiment, the Fc is human IgG4 E233F234L235to P233V234A235 with deletion G236 (IgG4Δb). In another embodiment, theFc is any human IgG4 Fc (IgG4, IgG4Δb or IgG4Δc) containing hingestabilizing mutation S228 to P228 (Aalberse et al., Immunology 105,9-19, 2002). In another embodiment, the Fc can be aglycosylated Fc.

In some embodiments, the constant region is aglycosylated by mutatingthe oligosaccharide attachment residue (such as Asn297) and/or flankingresidues that are part of the glycosylation recognition sequence in theconstant region. In some embodiments, the constant region isaglycosylated for N-linked glycosylation enzymatically. The constantregion may be aglycosylated for N-linked glycosylation enzymatically orby expression in a glycosylation deficient host cell.

In some embodiments, the constant region has a modified constant regionthat removes or reduces Fc gamma receptor binding. For example, the Fccan be human IgG2 containing the mutation D265, in which the amino acidresidues are numbered with reference to the wild type IgG2 sequence (SEQID NO: 493). Accordingly, in some embodiments, the constant region has amodified constant region having the sequence shown in SEQ ID NO: 496:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCRVRCPRCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNVVYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments, the constant region has a modified constant regionhaving the sequence shown in SEQ ID NO: 497:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNVVYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

One way of determining binding affinity of antibodies to BCMA or CD3 isby measuring binding affinity of monofunctional Fab fragments of theantibody. To obtain monofunctional Fab fragments, an antibody (forexample, IgG) can be cleaved with papain or expressed recombinantly. Theaffinity of a BCMA Fab fragment of an antibody can be determined bysurface plasmon resonance (Biacore™3000™ surface plasmon resonance (SPR)system, Biacore™, INC, Piscataway N.J.) equipped with pre-immobilizedstreptavidin sensor chips (SA) or anti-mouse Fc or anti-human Fc usingHBS-EP running buffer (0.01M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005%v/v Surfactant P20). Biotinylated or Fc fusion human BCMA can be dilutedinto HBS-EP buffer to a concentration of less than 0.5 μg/mL andinjected across the individual chip channels using variable contacttimes, to achieve two ranges of antigen density, either 50-200 responseunits (RU) for detailed kinetic studies or 800-1,000 RU for screeningassays. Regeneration studies have shown that 25 mM NaOH in 25% v/vethanol effectively removes the bound Fab while keeping the activity ofBCMA on the chip for over 200 injections. Typically, serial dilutions(spanning concentrations of 0.1-10× estimated K_(D)) of purified Fabsamples are injected for 1 min at 100 μL/minute and dissociation timesof up to 2 hours are allowed. The concentrations of the Fab proteins aredetermined by ELISA and/or SDS-PAGE electrophoresis using a Fab of knownconcentration (as determined by amino acid analysis) as a standard.Kinetic association rates (k_(on)) and dissociation rates (k_(off)) areobtained simultaneously by fitting the data globally to a 1:1 Langmuirbinding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994).Methods Enzymology 6. 99-110) using the BIAevaluation program.Equilibrium dissociation constant (K_(D)) values are calculated ask_(off)/k_(on). This protocol is suitable for use in determining bindingaffinity of an antibody to any BCMA, including human BCMA, BCMA ofanother mammal (such as mouse BCMA, rat BCMA, or primate BCMA), as wellas different forms of BCMA (e.g., glycosylated BCMA). Binding affinityof an antibody is generally measured at 25° C., but can also be measuredat 37° C.

The antibodies as described herein may be made by any method known inthe art. For the production of hybridoma cell lines, the route andschedule of immunization of the host animal are generally in keepingwith established and conventional techniques for antibody stimulationand production, as further described herein. General techniques forproduction of human and mouse antibodies are known in the art and/or aredescribed herein.

It is contemplated that any mammalian subject including humans orantibody producing cells therefrom can be manipulated to serve as thebasis for production of mammalian, including human and hybridoma celllines. Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, and/orintradermally with an amount of immunogen, including as describedherein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C., Nature 256:495-497, 1975 or as modified by Buck, D.W., et al., In Vitro, 18:377-381, 1982. Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the monoclonal antibodies of the subject invention.The hybridomas are expanded and subcloned, if desired, and supernatantsare assayed for anti-immunogen activity by conventional immunoassayprocedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescenceimmunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies specific for BCMA, CD3, or portions thereof.

Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity, if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen. Immunization of a host animal with a human BCMA or CD3, or afragment containing the target amino acid sequence conjugated to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups, can yield a population ofantibodies (e.g., monoclonal antibodies).

If desired, the antibody (monoclonal or polyclonal) of interest may besequenced and the polynucleotide sequence may then be cloned into avector for expression or propagation. The sequence encoding the antibodyof interest may be maintained in vector in a host cell and the host cellcan then be expanded and frozen for future use. Production ofrecombinant monoclonal antibodies in cell culture can be carried outthrough cloning of antibody genes from B cells by means known in theart. See, e.g. Tiller et al., J. Immunol. Methods 329, 112, 2008; U.S.Pat. No. 7,314,622.

In an alternative, the polynucleotide sequence may be used for geneticmanipulation to “humanize” the antibody or to improve the affinity, orother characteristics of the antibody. For example, the constant regionmay be engineered to more nearly resemble human constant regions toavoid immune response if the antibody is used in clinical trials andtreatments in humans. It may be desirable to genetically manipulate theantibody sequence to obtain greater affinity to BCMA or CD3 and greaterefficacy in inhibiting BCMA.

There are four general steps to humanize a monoclonal antibody. Theseare: (1) determining the nucleotide and predicted amino acid sequence ofthe starting antibody light and heavy variable domains (2) designing thehumanized antibody, i.e., deciding which antibody framework region touse during the humanizing process (3) the actual humanizingmethodologies/techniques and (4) the transfection and expression of thehumanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;5,585,089; and 6,180,370.

A number of “humanized” antibody molecules comprising an antigen bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent or modified rodent V regionsand their associated CDRs fused to human constant regions. See, forexample, Winter et al. Nature 349:293-299, 1991, Lobuglio et al. Proc.Nat. Acad. Sci. USA 86:4220-4224, 1989, Shaw et al. J Immunol.138:4534-4538, 1987, and Brown et al. Cancer Res. 47:3577-3583, 1987.Other references describe rodent CDRs grafted into a human supportingframework region (FR) prior to fusion with an appropriate human antibodyconstant region. See, for example, Riechmann et al. Nature 332:323-327,1988, Verhoeyen et al. Science 239:1534-1536, 1988, and Jones et al.Nature 321:522-525, 1986. Another reference describes rodent CDRssupported by recombinantly engineered rodent framework regions. See, forexample, European Patent Publication No. 0519596. These “humanized”molecules are designed to minimize unwanted immunological responsetoward rodent anti-human antibody molecules which limits the durationand effectiveness of therapeutic applications of those moieties in humanrecipients. For example, the antibody constant region can be engineeredsuch that it is immunologically inert (e.g., does not trigger complementlysis). See, e.g. PCT Publication No. PCT/GB99/01441; UK PatentApplication No. 9809951.8. Other methods of humanizing antibodies thatmay also be utilized are disclosed by Daugherty et al., Nucl. Acids Res.19:2471-2476, 1991, and in U.S. Pat. Nos. 6,180,377; 6,054,297;5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT PublicationNo. WO 01/27160.

The general principles related to humanized antibodies discussed aboveare also applicable to customizing antibodies for use, for example, indogs, cats, primate, equines and bovines. Further, one or more aspectsof humanizing an antibody described herein may be combined, e.g., CDRgrafting, framework mutation and CDR mutation.

In one variation, fully human antibodies may be obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse™ fromAbgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.).

In an alternative, antibodies may be made recombinantly and expressedusing any method known in the art. In another alternative, antibodiesmay be made recombinantly by phage display technology. See, for example,U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; andWinter et al., Annu. Rev. Immunol. 12:433-455, 1994. Alternatively, thephage display technology (McCafferty et al., Nature 348:552-553, 1990)can be used to produce human antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B cell. Phage display can be performed in a variety offormats; for review see, e.g., Johnson, Kevin S. and Chiswell, David J.,Current Opinion in Structural Biology 3:564-571, 1993. Several sourcesof V-gene segments can be used for phage display. Clackson et al.,Nature 352:624-628, 1991, isolated a diverse array of anti-oxazoloneantibodies from a small random combinatorial library of V genes derivedfrom the spleens of immunized mice. A repertoire of V genes fromunimmunized human donors can be constructed and antibodies to a diversearray of antigens (including self-antigens) can be isolated essentiallyfollowing the techniques described by Mark et al., J. Mol. Biol.222:581-597, 1991, or Griffith et al., EMBO J. 12:725-734, 1993. In anatural immune response, antibody genes accumulate mutations at a highrate (somatic hypermutation). Some of the changes introduced will conferhigher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling.” (Marks et al.,Bio/Technol. 10:779-783, 1992). In this method, the affinity of“primary” human antibodies obtained by phage display can be improved bysequentially replacing the heavy and light chain V region genes withrepertoires of naturally occurring variants (repertoires) of V domaingenes obtained from unimmunized donors. This technique allows theproduction of antibodies and antibody fragments with affinities in thepM-nM range. A strategy for making very large phage antibody repertoires(also known as “the mother-of-all libraries”) has been described byWaterhouse et al., Nucl. Acids Res. 21:2265-2266, 1993. Gene shufflingcan also be used to derive human antibodies from rodent antibodies,where the human antibody has similar affinities and specificities to thestarting rodent antibody. According to this method, which is alsoreferred to as “epitope imprinting”, the heavy or light chain V domaingene of rodent antibodies obtained by phage display technique isreplaced with a repertoire of human V domain genes, creatingrodent-human chimeras. Selection on antigen results in isolation ofhuman variable regions capable of restoring a functional antigen bindingsite, i.e., the epitope governs (imprints) the choice of partner. Whenthe process is repeated in order to replace the remaining rodent Vdomain, a human antibody is obtained (see PCT Publication No. WO93/06213). Unlike traditional humanization of rodent antibodies by CDRgrafting, this technique provides completely human antibodies, whichhave no framework or CDR residues of rodent origin.

Antibodies may be made recombinantly by first isolating the antibodiesand antibody producing cells from host animals, obtaining the genesequence, and using the gene sequence to express the antibodyrecombinantly in host cells (e.g., CHO cells). Another method which maybe employed is to express the antibody sequence in plants (e.g.,tobacco) or transgenic milk. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters, et al. Vaccine 19:2756, 2001; Lonberg, N. and D. Huszar Int.Rev. Immunol 13:65, 1995; and Pollock, et al., J Immunol Methods231:147, 1999. Methods for making derivatives of antibodies, e.g.,humanized, single chain, etc. are known in the art.

Immunoassays and flow cytometry sorting techniques such as fluorescenceactivated cell sorting (FACS) can also be employed to isolate antibodiesthat are specific for BCMA, CD3, or tumor antigens of interest.

The antibodies as described herein can be bound to many differentcarriers. Carriers can be active and/or inert. Examples of well-knowncarriers include polypropylene, polystyrene, polyethylene, dextran,nylon, amylases, glass, natural and modified celluloses,polyacrylamides, agaroses, and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingantibodies, or will be able to ascertain such, using routineexperimentation. In some embodiments, the carrier comprises a moietythat targets the myocardium.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors (such as expression vectors disclosed in PCTPublication No. WO 87/04462), which are then transfected into host cellssuch as E. coli cells, simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. See, e.g., PCT Publication No. WO 87/04462. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant regions in place ofthe homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci.81:6851, 1984, or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In that manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of a monoclonal antibodyherein.

The BCMA or tumor antigen of interest antibodies as described herein canbe identified or characterized using methods known in the art, wherebyreduction of BCMA or other tumor antigen expression levels are detectedand/or measured. In some embodiments, a BCMA antibody is identified byincubating a candidate agent with BCMA and monitoring binding and/orattendant reduction of BCMA expression levels. The binding assay may beperformed with purified BCMA polypeptide(s), or with cells naturallyexpressing, or transfected to express, BCMA polypeptide(s). In oneembodiment, the binding assay is a competitive binding assay, where theability of a candidate antibody to compete with a known BCMA antibodyfor BCMA binding is evaluated. The assay may be performed in variousformats, including the ELISA format.

Following initial identification, the activity of a candidate BCMA, CD3,or other tumor antigen antibody can be further confirmed and refined bybioassays, known to test the targeted biological activities.Alternatively, bioassays can be used to screen candidates directly. Someof the methods for identifying and characterizing antibodies aredescribed in detail in the Examples.

BCMA, CD3, or other tumor antigen antibodies may be characterized usingmethods well known in the art. For example, one method is to identifythe epitope to which it binds, or “epitope mapping.” There are manymethods known in the art for mapping and characterizing the location ofepitopes on proteins, including solving the crystal structure of anantibody-antigen complex, competition assays, gene fragment expressionassays, and synthetic peptide-based assays, as described, for example,in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. Inan additional example, epitope mapping can be used to determine thesequence to which an antibody binds. Epitope mapping is commerciallyavailable from various sources, for example, Pepscan Systems(Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope can bea linear epitope, i.e., contained in a single stretch of amino acids, ora conformational epitope formed by a three-dimensional interaction ofamino acids that may not necessarily be contained in a single stretch.Peptides of varying lengths (e.g., at least 4-6 amino acids long) can beisolated or synthesized (e.g., recombinantly) and used for bindingassays with a BCMA, CD3, or other tumor antigen antibody. In anotherexample, the epitope to which the BCMA, CD3, or other tumor antigenantibody binds can be determined in a systematic screening by usingoverlapping peptides derived from the BCMA, CD3, or other tumor antigensequence and determining binding by the BCMA, CD3, or other tumorantigen antibody. According to the gene fragment expression assays, theopen reading frame encoding BCMA, CD3, or other tumor antigen isfragmented either randomly or by specific genetic constructions and thereactivity of the expressed fragments of BCMA, CD3, or other tumorantigen with the antibody to be tested is determined. The gene fragmentsmay, for example, be produced by PCR and then transcribed and translatedinto protein in vitro, in the presence of radioactive amino acids. Thebinding of the antibody to the radioactively labeled BCMA, CD3, or othertumor antigen fragments is then determined by immunoprecipitation andgel electrophoresis. Certain epitopes can also be identified by usinglarge libraries of random peptide sequences displayed on the surface ofphage particles (phage libraries). Alternatively, a defined library ofoverlapping peptide fragments can be tested for binding to the testantibody in simple binding assays. In an additional example, mutagenesisof an antigen binding domain, domain swapping experiments and alaninescanning mutagenesis can be performed to identify residues required,sufficient, and/or necessary for epitope binding. For example, domainswapping experiments can be performed using a mutant BCMA, CD3, or othertumor antigen in which various fragments of the BCMA, CD3, or othertumor antigen protein have been replaced (swapped) with sequences fromBCMA from another species (e.g., mouse), or a closely related, butantigenically distinct protein (e.g., Trop-1). By assessing binding ofthe antibody to the mutant BCMA, CD3, or other tumor antigen, theimportance of the particular BCMA, CD3, or other tumor antigen fragmentto antibody binding can be assessed.

Yet another method which can be used to characterize a BCMA, CD3, orother tumor antigen antibody is to use competition assays with otherantibodies known to bind to the same antigen, i.e., various fragments onBCMA, CD3, or other tumor antigen, to determine if the BCMA, CD3, orother tumor antigen antibody binds to the same epitope as otherantibodies. Competition assays are well known to those of skill in theart.

An expression vector can be used to direct expression of a BCMA, CD3, orother tumor antigen antibody. One skilled in the art is familiar withadministration of expression vectors to obtain expression of anexogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908;6,413,942; and 6,376,471. Administration of expression vectors includeslocal or systemic administration, including injection, oraladministration, particle gun or catheterized administration, and topicaladministration. In another embodiment, the expression vector isadministered directly to the sympathetic trunk or ganglion, or into acoronary artery, atrium, ventrical, or pericardium.

Targeted delivery of therapeutic compositions containing an expressionvector, or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described in, for example,Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et al., GeneTherapeutics: Methods And Applications Of Direct Gene Transfer, J. A.Wolff, ed., 1994; Wu et al., J. Biol. Chem., 263:621, 1988; Wu et al.,J. Biol. Chem., 269:542, 1994; Zenke et al., Proc. Natl. Acad. Sci. USA,87:3655, 1990; and Wu et al., J. Biol. Chem., 266:338, 1991. Therapeuticcompositions containing a polynucleotide are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. Concentration ranges of about 500 ng to about 50 mg,about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 toabout 100 μg of DNA can also be used during a gene therapy protocol. Thetherapeutic polynucleotides and polypeptides can be delivered using genedelivery vehicles. The gene delivery vehicle can be of viral ornon-viral origin (see generally, Jolly, Cancer Gene Therapy, 1:51, 1994;Kimura, Human Gene Therapy, 5:845, 1994; Connelly, Human Gene Therapy,1995, 1:185; and Kaplitt, Nature Genetics, 6:148, 1994). Expression ofsuch coding sequences can be induced using endogenous mammalian orheterologous promoters. Expression of the coding sequence can be eitherconstitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Pat. No. 2,200,651; and EP Pat.No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors,Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCCVR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCCVR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associatedvirus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655).Administration of DNA linked to killed adenovirus as described inCuriel, Hum. Gene Ther., 1992, 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther., 3:147,1992); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem., 264:16985,1989); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No.5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763;and WO 97/42338) and nucleic charge neutralization or fusion with cellmembranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additionalapproaches are described in Philip, Mol. Cell Biol., 14:2411, 1994 andin Woffendin, Proc. Natl. Acad. Sci., 91:1581, 1994.

In some embodiments, the invention encompasses compositions, includingpharmaceutical compositions, comprising antibodies described herein ormade by the methods and having the characteristics described herein. Asused herein, compositions comprise one or more antibodies that bind toCD3 and a tumor antigen (e.g BCMA), and/or one or more polynucleotidescomprising sequences encoding one or more these antibodies. Thesecompositions may further comprise suitable excipients, such aspharmaceutically acceptable excipients including buffers, which are wellknown in the art.

The invention also provides methods of making any of these antibodies.The antibodies of this invention can be made by procedures known in theart. The polypeptides can be produced by proteolytic or otherdegradation of the antibodies, by recombinant methods (i.e., single orfusion polypeptides) as described above or by chemical synthesis.Polypeptides of the antibodies, especially shorter polypeptides up toabout 50 amino acids, are conveniently made by chemical synthesis.Methods of chemical synthesis are known in the art and are commerciallyavailable. For example, an antibody could be produced by an automatedpolypeptide synthesizer employing the solid phase method. See also, U.S.Pat. Nos. 5,807,715; 4,816,567; and 6,331,415.

Heteroconjugate antibodies, comprising two covalently joined antibodies,are also within the scope of the invention. Such antibodies have beenused to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (PCT Publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents and techniques are well known in the art, and are described inU.S. Pat. No. 4,676,980.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods of synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

In the recombinant humanized antibodies, the Fcγ portion can be modifiedto avoid interaction with Fcγ receptor and the complement and immunesystems. The techniques for preparation of such antibodies are describedin WO 99/58572. For example, the constant region may be engineered tomore resemble human constant regions to avoid immune response if theantibody is used in clinical trials and treatments in humans. See, forexample, U.S. Pat. Nos. 5,997,867 and 5,866,692.

The invention encompasses modifications to the antibodies andpolypeptides of the invention variants as described herein, includingfunctionally equivalent antibodies which do not significantly affecttheir properties and variants which have enhanced or decreased activityand/or affinity. For example, the amino acid sequence may be mutated toobtain an antibody with the desired binding affinity to BCMA and/or CD3.Modification of polypeptides is routine practice in the art and need notbe described in detail herein. Examples of modified polypeptides includepolypeptides with conservative substitutions of amino acid residues, oneor more deletions or additions of amino acids which do not significantlydeleteriously change the functional activity, or which mature (enhance)the affinity of the polypeptide for its ligand, or use of chemicalanalogs.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the half-life of the antibody in theblood circulation.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 5 under the heading of“conservative substitutions.” If such substitutions result in a changein biological activity, then more substantial changes, denominated“exemplary substitutions” in Table 5, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 5 Amino Acid Substitutions Original Residue (naturally occurringamino Conservative acid) Substitutions Exemplary Substitutions Ala (A)Val Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp,Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; GluGlu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile(I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile;Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; IlePhe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr(T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V)Leu Ile; Leu; Met; Phe; Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring amino acid residues are divided into groups based on commonside-chain properties:

-   -   (1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) Polar without charge: Cys, Ser, Thr, Asn, Gin;    -   (3) Acidic (negatively charged): Asp, Glu;    -   (4) Basic (positively charged): Lys, Arg;    -   (5) Residues that influence chain orientation: Gly, Pro; and    -   (6) Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are made by exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcross-linking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability, particularly where the antibody is an antibodyfragment such as an Fv fragment.

Amino acid modifications can range from changing or modifying one ormore amino acids to complete redesign of a region, such as the variableregion. Changes in the variable region can alter binding affinity and/orspecificity. In some embodiments, no more than one to five conservativeamino acid substitutions are made within a CDR domain. In otherembodiments, no more than one to three conservative amino acidsubstitutions are made within a CDR domain. In still other embodiments,the CDR domain is CDR H3 and/or CDR L3.

Modifications also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Antibodies are glycosylated atconserved positions in their constant regions (Jefferis and Lund, Chem.Immunol. 65:111-128, 1997; Wright and Morrison, TibTECH 15:26-32, 1997).The oligosaccharide side chains of the immunoglobulins affect theprotein's function (Boyd et al., Mol. Immunol. 32:1311-1318, 1996;Wittwe and Howard, Biochem. 29:4175-4180, 1990) and the intramolecularinteraction between portions of the glycoprotein, which can affect theconformation and presented three-dimensional surface of the glycoprotein(Jefferis and Lund, supra; Wyss and Wagner, Current Opin. Biotech.7:409-416, 1996). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al., Mature Biotech.17:176-180, 1999).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of antibodies may also be altered withoutaltering the underlying nucleotide sequence. Glycosylation largelydepends on the host cell used to express the antibody. Since the celltype used for expression of recombinant glycoproteins, e.g. antibodies,as potential therapeutics is rarely the native cell, variations in theglycosylation pattern of the antibodies can be expected (see, e.g. Hseet al., J. Biol. Chem. 272:9062-9070, 1997).

In addition to the choice of host cells, factors that affectglycosylation during recombinant production of antibodies include growthmode, media formulation, culture density, oxygenation, pH, purificationschemes and the like. Various methods have been proposed to alter theglycosylation pattern achieved in a particular host organism includingintroducing or overexpressing certain enzymes involved inoligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and5,278,299). Glycosylation, or certain types of glycosylation, can beenzymatically removed from the glycoprotein, for example, usingendoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1,endoglycosidase F2, endoglycosidase F3. In addition, the recombinanthost cell can be genetically engineered to be defective in processingcertain types of polysaccharides. These and similar techniques are wellknown in the art.

Other methods of modification include using coupling techniques known inthe art, including, but not limited to, enzymatic means, oxidativesubstitution and chelation. Modifications can be used, for example, forattachment of labels for immunoassay. Modified polypeptides are madeusing established procedures in the art and can be screened usingstandard assays known in the art, some of which are described below andin the Examples.

In some embodiments of the invention, the antibody comprises a modifiedconstant region, such as a constant region that has increased affinityto a human Fc gamma receptor, is immunologically inert or partiallyinert, e.g., does not trigger complement mediated lysis, does notstimulate antibody-dependent cell mediated cytotoxicity (ADCC), or doesnot activate macrophages; or has reduced activities (compared to theunmodified antibody) in any one or more of the following: triggeringcomplement mediated lysis, stimulating antibody-dependent cell mediatedcytotoxicity (ADCC), or activating microglia. Different modifications ofthe constant region may be used to achieve optimal level and/orcombination of effector functions. See, for example, Morgan et al.,Immunology 86:319-324, 1995; Lund et al., J. Immunology 157:4963-9157:4963-4969, 1996; Idusogie et al., J. Immunology 164:4178-4184, 2000;Tao et al., J. Immunology 143: 2595-2601, 1989; and Jefferis et al.,Immunological Reviews 163:59-76, 1998. In some embodiments, the constantregion is modified as described in Eur. J. Immunol., 1999, 29:2613-2624;PCT Application No. PCT/GB99/01441; and/or UK Patent Application No.9809951.8. In other embodiments, the antibody comprises a human heavychain IgG2 constant region comprising the following mutations: A330P331to S330S331 (amino acid numbering with reference to the wild type IgG2sequence). Eur. J. Immunol., 1999, 29:2613-2624. In still otherembodiments, the constant region is aglycosylated for N-linkedglycosylation. In some embodiments, the constant region is aglycosylatedfor N-linked glycosylation by mutating the glycosylated amino acidresidue or flanking residues that are part of the N-glycosylationrecognition sequence in the constant region. For example,N-glycosylation site N297 may be mutated to A, Q, K, or H. See, Tao etal., J. Immunology 143: 2595-2601, 1989; and Jefferis et al.,Immunological Reviews 163:59-76, 1998. In some embodiments, the constantregion is aglycosylated for N-linked glycosylation. The constant regionmay be aglycosylated for N-linked glycosylation enzymatically (such asremoving carbohydrate by enzyme PNGase), or by expression in aglycosylation deficient host cell.

Other antibody modifications include antibodies that have been modifiedas described in PCT Publication No. WO 99/58572. These antibodiescomprise, in addition to a binding domain directed at the targetmolecule, an effector domain having an amino acid sequence substantiallyhomologous to all or part of a constant region of a human immunoglobulinheavy chain. These antibodies are capable of binding the target moleculewithout triggering significant complement dependent lysis, orcell-mediated destruction of the target. In some embodiments, theeffector domain is capable of specifically binding FcRn and/or FcγRIIb.These are typically based on chimeric domains derived from two or morehuman immunoglobulin heavy chain C_(H)2 domains. Antibodies modified inthis manner are particularly suitable for use in chronic antibodytherapy, to avoid inflammatory and other adverse reactions toconventional antibody therapy.

The invention includes affinity matured embodiments. For example,affinity matured antibodies can be produced by procedures known in theart (Marks et al., Bio/Technology, 10:779-783, 1992; Barbas et al., ProcNat. Acad. Sci, USA 91:3809-3813, 1994; Schier et al., Gene,169:147-155, 1995; Yelton et al., J. Immunol., 155:1994-2004, 1995;Jackson et al., J. Immunol., 154(7):3310-9, 1995, Hawkins et al., J.Mol. Biol., 226:889-896, 1992; and PCT Publication No. WO2004/058184).

The following methods may be used for adjusting the affinity of anantibody and for characterizing a CDR. One way of characterizing a CDRof an antibody and/or altering (such as improving) the binding affinityof a polypeptide, such as an antibody, termed “library scanningmutagenesis”. Generally, library scanning mutagenesis works as follows.One or more amino acid positions in the CDR are replaced with two ormore (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20) amino acids using art recognized methods. This generatessmall libraries of clones (in some embodiments, one for every amino acidposition that is analyzed), each with a complexity of two or moremembers (if two or more amino acids are substituted at every position).Generally, the library also includes a clone comprising the native(unsubstituted) amino acid. A small number of clones, e.g., about 20-80clones (depending on the complexity of the library), from each libraryare screened for binding affinity to the target polypeptide (or otherbinding target), and candidates with increased, the same, decreased, orno binding are identified. Methods for determining binding affinity arewell-known in the art. Binding affinity may be determined using Biacore™surface plasmon resonance analysis, which detects differences in bindingaffinity of about 2-fold or greater. Biacore™ is particularly usefulwhen the starting antibody already binds with a relatively highaffinity, for example a K_(D) of about 10 nM or lower. Screening usingBiacore™ surface plasmon resonance is described in the Examples, herein.

Binding affinity may be determined using Kinexa Biocensor, scintillationproximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescencequenching, fluorescence transfer, and/or yeast display. Binding affinitymay also be screened using a suitable bioassay.

In some embodiments, every amino acid position in a CDR is replaced (insome embodiments, one at a time) with all 20 natural amino acids usingart recognized mutagenesis methods (some of which are described herein).This generates small libraries of clones (in some embodiments, one forevery amino acid position that is analyzed), each with a complexity of20 members (if all 20 amino acids are substituted at every position).

In some embodiments, the library to be screened comprises substitutionsin two or more positions, which may be in the same CDR or in two or moreCDRs. Thus, the library may comprise substitutions in two or morepositions in one CDR. The library may comprise substitution in two ormore positions in two or more CDRs. The library may comprisesubstitution in 3, 4, 5, or more positions, said positions found in two,three, four, five or six CDRs. The substitution may be prepared usinglow redundancy codons. See, e.g., Table 2 of Balint et al., Gene137(1):109-18, 1993.

The CDR may be CDRH3 and/or CDRL3. The CDR may be one or more of CDRL1,CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH3. The CDR may be a Kabat CDR, aChothia CDR, or an extended CDR.

Candidates with improved binding may be sequenced, thereby identifying aCDR substitution mutant which results in improved affinity (also termedan “improved” substitution). Candidates that bind may also be sequenced,thereby identifying a CDR substitution which retains binding.

Multiple rounds of screening may be conducted. For example, candidates(each comprising an amino acid substitution at one or more position ofone or more CDR) with improved binding are also useful for the design ofa second library containing at least the original and substituted aminoacid at each improved CDR position (i.e., amino acid position in the CDRat which a substitution mutant showed improved binding). Preparation,and screening or selection of this library is discussed further below.

Library scanning mutagenesis also provides a means for characterizing aCDR, in so far as the frequency of clones with improved binding, thesame binding, decreased binding or no binding also provide informationrelating to the importance of each amino acid position for the stabilityof the antibody-antigen complex. For example, if a position of the CDRretains binding when changed to all 20 amino acids, that position isidentified as a position that is unlikely to be required for antigenbinding. Conversely, if a position of CDR retains binding in only asmall percentage of substitutions, that position is identified as aposition that is important to CDR function. Thus, the library scanningmutagenesis methods generate information regarding positions in the CDRsthat can be changed to many different amino acids (including all 20amino acids), and positions in the CDRs which cannot be changed or whichcan only be changed to a few amino acids.

Candidates with improved affinity may be combined in a second library,which includes the improved amino acid, the original amino acid at thatposition, and may further include additional substitutions at thatposition, depending on the complexity of the library that is desired, orpermitted using the desired screening or selection method. In addition,if desired, adjacent amino acid position can be randomized to at leasttwo or more amino acids. Randomization of adjacent amino acids maypermit additional conformational flexibility in the mutant CDR, whichmay in turn, permit or facilitate the introduction of a larger number ofimproving mutations. The library may also comprise substitution atpositions that did not show improved affinity in the first round ofscreening.

The second library is screened or selected for library members withimproved and/or altered binding affinity using any method known in theart, including screening using Biacore™ surface plasmon resonanceanalysis, and selection using any method known in the art for selection,including phage display, yeast display, and ribosome display.

This invention also provides compositions comprising antibodiesconjugated (for example, linked) to an agent that facilitate coupling toa solid support (such as biotin or avidin). For simplicity, referencewill be made generally to antibodies with the understanding that thesemethods apply to any of the BCMA antibody embodiments described herein.Conjugation generally refers to linking these components as describedherein. The linking (which is generally fixing these components inproximate association at least for administration) can be achieved inany number of ways. For example, a direct reaction between an agent andan antibody is possible when each possesses a substituent capable ofreacting with the other. For example, a nucleophilic group, such as anamino or sulfhydryl group, on one may be capable of reacting with acarbonyl-containing group, such as an anhydride or an acid halide, orwith an alkyl group containing a good leaving group (e.g., a halide) onthe other.

In another aspect, the invention provides a method of making any of thepolynucleotides described herein.

Polynucleotides complementarity to any such sequences are alsoencompassed by the present invention. Polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. RNA molecules includeHnRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mRNA molecules, which do not containintrons. Additional coding or non-coding sequences may, but need not, bepresent within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an antibody or a portion thereof) or may comprisea variant of such a sequence. Polynucleotide variants contain one ormore substitutions, additions, deletions and/or insertions such that theimmunoreactivity of the encoded polypeptide is not diminished, relativeto a native immunoreactive molecule. The effect on the immunoreactivityof the encoded polypeptide may generally be assessed as describedherein. Variants preferably exhibit at least about 70% identity, morepreferably, at least about 80% identity, yet more preferably, at leastabout 90% identity, and most preferably, at least about 95% identity toa polynucleotide sequence that encodes a native antibody or a portionthereof.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, or 40 to about 50, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O., 1978, A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e. the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a nativeantibody (or a complementarity sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

The polynucleotides of this invention can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al., 1989.

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., 1989, supra, for example.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a polynucleotide according to the invention. It is impliedthat an expression vector must be replicable in the host cells either asepisomes or as an integral part of the chromosomal DNA. Suitableexpression vectors include but are not limited to plasmids, viralvectors, including adenoviruses, adeno-associated viruses, retroviruses,cosmids, and expression vector(s) disclosed in PCT Publication No. WO87/04462. Vector components may generally include, but are not limitedto, one or more of the following: a signal sequence; an origin ofreplication; one or more marker genes; suitable transcriptionalcontrolling elements (such as promoters, enhancers and terminator). Forexpression (i.e., translation), one or more translational controllingelements are also usually required, such as ribosome binding sites,translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

The invention also provides host cells comprising any of thepolynucleotides described herein. Any host cells capable ofover-expressing heterologous DNAs can be used for the purpose ofisolating the genes encoding the antibody, polypeptide or protein ofinterest. Non-limiting examples of mammalian host cells include but notlimited to COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such asE. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; orK. lactis). Preferably, the host cells express the cDNAs at a level ofabout 5 fold higher, more preferably, 10 fold higher, even morepreferably, 20 fold higher than that of the corresponding endogenousantibody or protein of interest, if present, in the host cells.Screening the host cells for a specific binding to BCMA or an BCMAdomain (e.g., domains 1-4) is effected by an immunoassay or FACS. A celloverexpressing the antibody or protein of interest can be identified.

Methods of Using the Bispecific Antibodies

The antibodies (e.g., BCMA, CD3, or bispecific) and the antibodyconjugates (e.g., BCMA antibody-drug conjugates) of the presentinvention are useful in various applications including, but are notlimited to, therapeutic treatment methods and diagnostic treatmentmethods.

In one aspect, the invention provides a method for treating a conditionassociated with BCMA expression in a subject. In some embodiments, themethod of treating a condition associated with BCMA expression in asubject comprises administering to the subject in need thereof aneffective amount of a composition (e.g., pharmaceutical composition)comprising the BCMA antibodies or the BCMA antibody conjugates asdescribed herein. The conditions associated with BCMA expressioninclude, but are not limited to, abnormal BCMA expression, altered oraberrant BCMA expression, malignant cells expressing BCMA, and aproliferative disorder (e.g., cancer) or autoimmune disorder.

In another aspect, the invention provides a method for treating a B-cellrelated cancer or malignant cells expressing a tumor antigen. In someembodiments, provided is a method of treating a B-cell related cancer ina subject in need thereof comprising a) providing the bispecificantibody as described herein, and b) administering said bispecificantibody to said patient. In some embodiments, provided is a method oftreating a condition associated with malignant cells expressing a tumorantigen in a subject comprising administering to the subject in needthereof an effective amount of a pharmaceutical composition comprisingthe bispecific antibody as described herein.

Accordingly, in some embodiments, provided is a method of treating acancer in a subject comprising administering to the subject in needthereof an effective amount of a composition comprising the antibodies(e.g., BCMA, or CD3-BCMA bispecific antibodies) or the BCMA antibodyconjugates as described herein. As used herein, cancer can be a B-cellrelated cancer including, but are not limited to, multiple myeloma,malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocytepredominant Hodgkin's lymphoma, Kahler's disease and Myelomatosis,plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia,hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloidleukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocyticleukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma,Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, largecell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia,Waldenstrom's macroglobulienemia, diffuse large B cell lymphoma,follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphatictissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma,Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma,lymphoplasmactyic lymphoma, Waldenström macroglobulinemia, nodalmarginal zone B cell lymphoma, splenic marginal zone lymphoma,intravascular large B-cell lymphoma, primary effusion lymphoma,lymphomatoid granulomatosis, T cell/histiocyte-rich large B-celllymphoma, primary central nervous system lymphoma, primary cutaneousdiffuse large B-cell lymphoma (leg type), EBV positive diffuse largeB-cell lymphoma of the elderly, diffuse large B-cell lymphoma associatedwith inflammation, intravascular large B-cell lymphoma, ALK-positivelarge B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphomaarising in HHV8-associated multicentric Castleman disease, B-celllymphoma unclassified with features intermediate between diffuse largeB-cell lymphoma and Burkitt lymphoma, B-cell lymphoma unclassified withfeatures intermediate between diffuse large B-cell lymphoma andclassical Hodgkin lymphoma, and other B-cell related lymphoma.

In some embodiments, provided is a method of inhibiting tumor growth orprogression in a subject who has malignant cells expressing BCMA,comprising administering to the subject in need thereof an effectiveamount of a composition comprising the BCMA antibodies, CD3-BCMAbispecific antibodies, or the BCMA antibody conjugates as describedherein. In other embodiments, provided is a method of inhibitingmetastasis cells expressing BCMA in a subject, comprising administeringto the subject in need thereof an effective amount of a compositioncomprising the BCMA antibodies, CD3-BCMA bispecific antibodies, or theBCMA antibody conjugates as described herein. In other embodiments,provided is a method of inducing tumor regression in malignant cells ina subject, comprising administering to the subject in need thereof aneffective amount of a composition comprising the BCMA antibodies,CD3-BCMA bispecific antibodies, or the BCMA antibody conjugates asdescribed herein.

In some embodiments, provided is a method of treating an autoimmunedisorder in a subject comprising administering to the subject in needthereof an effective amount of a composition comprising the BCMAantibodies, CD3-BCMA bispecific antibodies, or the BCMA antibodyconjugates as described herein.

As used herein, autoimmune disorders include, but are not limited to,systemic lupus erythematosus, rheumatoid arthritis, diabetes (Type I),multiple sclerosis, Addison's disease, celiac disease, dermatomyositis,Graves' disease, hashimoto's thyroiditis, hashimoto's encephalopathy,Myasthenia gravis, pernicious anemia, reactive arthritis, Sjogrensyndrome, acute disseminated encephalomyelitis, agammaglobulinemia,amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipidsyndrome, antisynthetase syndrome, atopic allergy, atopic dermatitis,autoimmune enteropathy, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune inner ear disease, autoimmune lymphoproliferativesyndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis,autoimmune polyendorcrine syndrome, autoimmune progesterone dermatitis,autoimmune thrombocytopenic purpura, autoimmune urticarial, autoimmuneuveitis, Bechet's disease, Castleman's disease, cold agglutinin disease,Crohn's disease, dermatomyositis, eosinophilic fasciitis,gastrointestinal pemphigoid, Goodpasture's syndrome, Guillain-Barrésyndrome, hidradenitis suppurativa, idiopathic thrombocytopenic purpura,narcolepsy, pemphigus vulgaris, pernicious anaemia, polymyositis,primary binary cirrhosis, relapsing polychrondritis, rheumatic fever,temporal arteritis, transverse myelitis, ulcerative colitis,undifferentiated connective tissue disease, vasculitis, and Wegener'sgranulomatosis.

In another aspect, the invention provides an effective amount of acomposition (e.g., pharmaceutical composition) comprising the antibodies(e.g., BCMA or CD3-BCMA bispecific) or the BCMA antibody conjugates asdescribed herein for treating a condition (e.g., cancer or autoimmunedisorder) associated with BCMA expression in a subject in need thereof.In some embodiments, provided is an effective amount of a composition(e.g., pharmaceutical composition) comprising the antibodies (e.g., BCMAor CD3-BCMA bispecific) or the BCMA antibody conjugates as describedherein for inhibiting tumor growth or progression in a subject who hasmalignant cells expressing BCMA. In some embodiments, provided is aneffective amount of a composition (e.g., pharmaceutical composition)comprising the antibodies (e.g., BCMA or CD3-BCMA bispecific) or theBCMA antibody conjugates as described herein for inhibiting metastasisof malignant cells expressing BCMA in a subject in need thereof. In someembodiments, provided is an effective amount of a composition (e.g.,pharmaceutical composition) comprising the antibodies (e.g., BCMA orCD3-BCMA bispecific) or the BCMA antibody conjugates as described hereinfor inducing tumor regression in a subject who has malignant cellsexpressing BCMA.

In another aspect, the invention provides the antibodies (e.g., BCMA orCD3-BCMA bispecific) or the BCMA antibody conjugates as described hereinfor use in treating a condition (e.g., cancer or autoimmune disorder)associated with BCMA expression in a subject in need thereof. In someembodiments, provided is the antibodies (e.g., BCMA or CD3-BCMAbispecific) or the BCMA antibody conjugates as described herein forinhibiting tumor growth or progression in a subject who has malignantcells expressing BCMA. In some embodiments, provided is the antibodies(e.g., BCMA or CD3-BCMA bispecific) or the BCMA antibody conjugates asdescribed herein for inhibiting metastasis of malignant cells expressingBCMA in a subject in need thereof. In some embodiments, provided is theantibodies (e.g., BCMA or CD3-BCMA bispecific) or the BCMA antibodyconjugates as described herein for inducing tumor regression in asubject who has malignant cells expressing BCMA.

In another aspect, the invention provides a use of the antibodies (e.g.,BCMA or CD3-BCMA bispecific) or the BCMA antibody conjugates asdescribed herein in the manufacture of a medicament for treating acondition (e.g., cancer or autoimmune disorder) associated with BCMAexpression. In some embodiments, provided is a use of the antibodies(e.g., BCMA or CD3-BCMA bispecific) or the BCMA antibody conjugates asdescribed herein in the manufacture of a medicament for inhibiting tumorgrowth or progression. In some embodiments, provided is a use of theantibodies (e.g., BCMA or CD3-BCMA bispecific) or the BCMA antibodyconjugates as described herein in the manufacture of a medicament forinhibiting metastasis of malignant cells expressing BCMA. In someembodiments, provided is a use of the antibodies (e.g., BCMA or CD3-BCMAbispecific) or the BCMA antibody conjugates as described herein in themanufacture of a medicament for inducing tumor regression.

In another aspect, provided is a method of detecting, diagnosing, and/ormonitoring a condition associated with BCMA expression. For example, theantibodies (e.g., BCMA or CD3-BCMA bispecific) as described herein canbe labeled with a detectable moiety such as an imaging agent and anenzyme-substrate label. The antibodies as described herein can also beused for in vivo diagnostic assays, such as in vivo imaging (e.g., PETor SPECT), or a staining reagent.

In some embodiments, the methods described herein further comprise astep of treating a subject with an additional form of therapy. In someembodiments, the additional form of therapy is an additional anti-cancertherapy including, but not limited to, chemotherapy, radiation, surgery,hormone therapy, and/or additional immunotherapy.

In some embodiments, the additional form of therapy comprisesadministering one or more therapeutic agent in addition to theantibodies (e.g., BCMA or CD3-BCMA bispecific) or the BCMA antibodyconjugates as described herein. The one or more therapeutic agent can bea chemotherapeutic agents including, but not limited to, a secondantibody (e.g., an anti-VEGF (Vascular Endothelial Growth Factor)antibody (e.g., AVASTIN®), an anti-HER2 antibody (e.g., HERCEPTIN®), ananti-CD25 antibody, an anti-CD33 antibody, an anti-CD20 antibody (e.g.,RITUXAN®), an anti-mucin-like glycoprotein antibody, an anti-TNFantibody, and/or an epidermal growth factor receptor (EGFR) antibody(e.g., ERBITUX®)), an angiogenesis inhibitor, a cytotoxic agent (e.g.,anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin,valrubicin, and mitoxantrone), taxane (e.g., paclitaxel and docetaxel),dolastatin, duocarmycin, enediyne, geldanamycin, maytansine, puromycin,vinca alkaloid (e.g., vincristine), a topoisomerase inhibitor (e.g.,etoposide), tubulysin, a pyrimidine analog (e.g., fluorouracil),platinum-containing agents (e.g., cisplatin, carboplatin, andoxaliplatin), alkylating agents (e.g., melphalan, cyclophosphamide, orcarmustine) and hemiasterlin), immunomodulating agent (e.g., prednisoneand lenalidomide (REVLIMID®)), an anti-inflammatory agent (e.g.,dexamethasone), an aromatase inhibitor (e.g., anastrozole, exemestane,letrozole, vorozole, formestane, or testolactone), a proteasomeinhibitor (e.g., bortezomib such as VELCADE®([(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propy-l]amino]butyl]boronic acid or carfilzomib), and other agents such as tamoxifen.

For example, in some embodiments, provided is a method of treatingmultiple myeloma comprising administering to a patient need thereof aneffective amount of a composition comprising the antibodies (e.g., BCMAor CD3-BCMA bispecific) or the BCMA antibody conjugates as describedherein and one or more other therapeutic agent such as achemotherapeutic agent (e.g., doxorubicin or carfilzomib) or thalidomideor its derivative thereof (e.g., lenalidomide (REVLIMID®)). In someembodiments, the one or more other therapeutic agent is selecting fromthe group consisting of bortezomib (e.g., VELCADE®), melphalan,prednisone, doxorubicin, lenalidomide, thalidomide, prednisone,carmustine, etoposide, cisplatin, cyclophosphamide, carfilzomib, andvincristine. In some embodiments, the other therapeutic agent isbortezomib (e.g., VELCADE®), melphalan, lenalidomide (REVLIMID®),carfilzomib, doxorubicin, or prednisone. Accordingly, provided is amethod of treating multiple myeloma comprising administering to apatient need thereof an effective amount of a composition comprising theantibodies (e.g., BCMA or CD3-BCMA bispecific) or the BCMA antibodyconjugates as described herein and one or more other therapeutic agentselecting from the group consisting of bortezomib, lenalidomide,carfilzomib, and doxorubicin. In some embodiments, the patient isrelapsing or refractory to previous multiple myeloma therapy.

The antibodies (e.g., BCMA or CD3-BCMA bispecific) or the BCMA antibodyconjugates can be administered to an individual via any suitable route.It should be understood by persons skilled in the art that the examplesdescribed herein are not intended to be limiting but to be illustrativeof the techniques available. Accordingly, in some embodiments, theantibody (e.g., BCMA or CD3-BCMA bispecific) or the BCMA antibodyconjugate is administered to an individual in accord with known methods,such as intravenous administration, e.g., as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerebrospinal, intracranial, transdermal, subcutaneous,intra-articular, sublingually, intrasynovial, via insufflation,intrathecal, oral, inhalation or topical routes. Administration can besystemic, e.g., intravenous administration, or localized. Commerciallyavailable nebulizers for liquid formulations, including jet nebulizersand ultrasonic nebulizers are useful for administration. Liquidformulations can be directly nebulized and lyophilized powder can benebulized after reconstitution. Alternatively, the antibody (e.g., BCMAor CD3-BCMA bispecific) or the BCMA antibody conjugate can beaerosolized using a fluorocarbon formulation and a metered dose inhaler,or inhaled as a lyophilized and milled powder.

In one embodiment, the antibody (e.g., BCMA or CD3-BCMA bispecific) orthe BCMA antibody conjugate is administered via site-specific ortargeted local delivery techniques. Examples of site-specific ortargeted local delivery techniques include various implantable depotsources of the antibody (e.g., BCMA or CD3-BCMA bispecific) or the BCMAantibody conjugate or local delivery catheters, such as infusioncatheters, indwelling catheters, or needle catheters, synthetic grafts,adventitial wraps, shunts and stents or other implantable devices, sitespecific carriers, direct injection, or direct application. See, e.g.,PCT Publication No. WO 00/53211 and U.S. Pat. No. 5,981,568.

Various formulations of the antibody (e.g., BCMA or CD3-BCMA bispecific)or the BCMA antibody conjugate may be used for administration. In someembodiments, the antibody (e.g., BCMA or CD3-BCMA bispecific) or theBCMA antibody conjugate may be administered neat. In some embodiments,the antibody (e.g., BCMA or CD3-BCMA bispecific) or the BCMA antibodyconjugate and a pharmaceutically acceptable excipient may be in variousformulations. Pharmaceutically acceptable excipients are known in theart, and are relatively inert substances that facilitate administrationof a pharmacologically effective substance. For example, an excipientcan give form or consistency, or act as a diluent. Suitable excipientsinclude but are not limited to stabilizing agents, wetting andemulsifying agents, salts for varying osmolarity, encapsulating agents,buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 21st Ed. MackPublishing, 2005.

In some embodiments, these agents are formulated for administration byinjection (e.g., intraperitoneally, intravenously, subcutaneously,intramuscularly, etc.). Accordingly, these agents can be combined withpharmaceutically acceptable vehicles such as saline, Ringer's solution,dextrose solution, and the like. The particular dosage regimen, i.e.,dose, timing and repetition, will depend on the particular individualand that individual's medical history.

The antibodies (e.g., BCMA or CD3-BCMA bispecific) or the BCMA antibodyconjugates as described herein can be administered using any suitablemethod, including by injection (e.g., intraperitoneally, intravenously,subcutaneously, intramuscularly, etc.). The antibody (e.g., BCMA orCD3-BCMA bispecific) or the BCMA antibody conjugate can also beadministered via inhalation, as described herein. Generally, foradministration of an antibody (e.g., BCMA or CD3-BCMA bispecific) and aBCMA antibody conjugate, an initial candidate dosage can be about 2mg/kg. For the purpose of the present invention, a typical daily dosagemight range from about any of 3 μg/kg to 30 μg/kg to 300 μg/kg to 3mg/kg, to 30 mg/kg, to 100 mg/kg or more, depending on the factorsmentioned above. For example, dosage of about 1 mg/kg, about 2.5 mg/kg,about 5 mg/kg, about 10 mg/kg, and about 25 mg/kg may be used. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved, forexample, to inhibit or delay tumor growth/progression or metatstasis ofcancer cells. An exemplary dosing regimen comprises administering aninitial dose of about 2 mg/kg, followed by a weekly maintenance dose ofabout 1 mg/kg of the antibody (e.g., BCMA or CD3-BCMA bispecific) orBCMA antibody conjugate, or followed by a maintenance dose of about 1mg/kg every other week. Other exemplary dosing regimen comprisesadministering increasing doses (e.g., initial dose of 1 mg/kg andgradual increase to one or more higher doses every week or longer timeperiod). Other dosage regimens may also be useful, depending on thepattern of pharmacokinetic decay that the practitioner wishes toachieve. For example, in some embodiments, dosing from one to four timesa week is contemplated. In other embodiments, dosing once a month oronce every other month or every three months is contemplated. Theprogress of this therapy is easily monitored by conventional techniquesand assays. The dosing regimen (including the antibody (e.g., BCMA orCD3-BCMA bispecific) or the BCMA antibody conjugate used) can vary overtime.

For the purpose of the present invention, the appropriate dosage of anantibody (e.g., BCMA or CD3-BCMA bispecific) or a BCMA antibodyconjugate will depend on the antibody (e.g., BCMA or CD3-BCMAbispecific) or the BCMA antibody conjugate (or compositions thereof)employed, the type and severity of symptoms to be treated, whether theagent is administered for therapeutic purposes, previous therapy, thepatient's clinical history and response to the agent, the patient'sclearance rate for the administered agent, and the discretion of theattending physician. Typically the clinician will administer an antibody(e.g., BCMA or CD3-BCMA bispecific) or a BCMA antibody conjugate until adosage is reached that achieves the desired result. Dose and/orfrequency can vary over course of treatment. Empirical considerations,such as the half-life, generally will contribute to the determination ofthe dosage. For example, antibodies that are compatible with the humanimmune system, such as humanized antibodies or fully human antibodies,may be used to prolong half-life of the antibody and to prevent theantibody being attacked by the host's immune system. Frequency ofadministration may be determined and adjusted over the course oftherapy, and is generally, but not necessarily, based on treatmentand/or suppression and/or amelioration and/or delay of symptoms, e.g.,tumor growth inhibition or delay, etc. Alternatively, sustainedcontinuous release formulations of antibodies (e.g., BCMA or CD3-BCMAbispecific) or BCMA antibody conjugates may be appropriate. Variousformulations and devices for achieving sustained release are known inthe art.

In one embodiment, dosages for an antibody (e.g., BCMA or CD3-BCMAbispecific) or a BCMA antibody conjugate may be determined empiricallyin individuals who have been given one or more administration(s) of theantibody (e.g., BCMA or CD3-BCMA bispecific) or the BCMA antibodyconjugate. Individuals are given incremental dosages of an antibody(e.g., BCMA or CD3-BCMA bispecific) or a BCMA antibody conjugate. Toassess efficacy, an indicator of the disease can be followed.

Administration of an antibody (e.g., BCMA or CD3-BCMA bispecific) or anBCMA antibody conjugate in accordance with the method in the presentinvention can be continuous or intermittent, depending, for example,upon the recipient's physiological condition, whether the purpose of theadministration is therapeutic or prophylactic, and other factors knownto skilled practitioners. The administration of an antibody (e.g., BCMAor CD3-BCMA bispecific) or a BCMA antibody conjugate may be essentiallycontinuous over a preselected period of time or may be in a series ofspaced doses.

In some embodiments, more than one antibody (e.g., BCMA or CD3-BCMAbispecific) or BCMA antibody conjugate may be present. At least one, atleast two, at least three, at least four, at least five different ormore antibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibodyconjugate can be present. Generally, those antibodies (e.g., BCMA orCD3-BCMA bispecific) or BCMA antibody conjugates may have complementaryactivities that do not adversely affect each other. For example, one ormore of the following antibody may be used: a first BCMA or CD3 antibodydirected to one epitope on BCMA or CD3 and a second BCMA or CD3 antibodydirected to a different epitope on BCMA or CD3.

Therapeutic formulations of the antibody (e.g., BCMA or CD3-BCMAbispecific) or the BCMA antibody conjugate used in accordance with thepresent invention are prepared for storage by mixing an antibody havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington, The Science and Practiceof Pharmacy 21st Ed. Mack Publishing, 2005), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and may comprise buffers such as phosphate, citrate, and otherorganic acids; salts such as sodium chloride; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Liposomes containing the antibody (e.g., BCMA or CD3-BCMA bispecific) orthe BCMA antibody conjugate are prepared by methods known in the art,such as described in Epstein, et al., Proc. Natl. Acad. Sci. USA82:3688, 1985; Hwang, et al., Proc. Natl Acad. Sci. USA 77:4030, 1980;and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556. Particularlyuseful liposomes can be generated by the reverse phase evaporationmethod with a lipid composition comprising phosphatidylcholine,cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).Liposomes are extruded through filters of defined pore size to yieldliposomes with the desired diameter.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington, The Science and Practice of Pharmacy 21st Ed. MackPublishing, 2005.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or ‘poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by, for example, filtration through sterilefiltration membranes. Therapeutic antibody (e.g., BCMA or CD3-BCMAbispecific) or BCMA antibody conjugate compositions are generally placedinto a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

The compositions according to the present invention may be in unitdosage forms such as tablets, pills, capsules, powders, granules,solutions or suspensions, or suppositories, for oral, parenteral orrectal administration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) andother sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g. egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing an antibody(e.g., BCMA or CD3-BCMA bispecific) or a BCMA antibody conjugate withIntralipid™ or the components thereof (soybean oil, egg phospholipids,glycerol and water).

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as set outabove. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

Compositions

The compositions used in the methods of the invention comprise aneffective amount of an antibody (e.g., BCMA or CD3-BCMA bispecific) or aBCMA antibody conjugate as described herein. Examples of suchcompositions, as well as how to formulate, are also described in anearlier section and below. In some embodiments, the compositioncomprises one or more antibodies (e.g., BCMA or CD3-BCMA bispecific) orBCMA antibody conjugates. For example, BCMA antibody or CD3-BCMAbispecific antibody recognizes human BCMA or CD3-BCMA. In someembodiments, the BCMA or CD3-BCMA antibody is a human antibody, ahumanized antibody, or a chimeric antibody. In some embodiments, theBCMA antibody or CD3-BCMA antibody comprises a constant region that iscapable of triggering a desired immune response, such asantibody-mediated lysis or ADCC. In other embodiments, the BCMA antibodyor CD3-BCMA antibody comprises a constant region that does not triggeran unwanted or undesirable immune response, such as antibody-mediatedlysis or ADCC.

It is understood that the compositions can comprise more than oneantibody (e.g., BCMA or CD3-BCMA bispecific) or BCMA antibody conjugate(e.g., a mixture of BCMA antibodies or CD3-BCMA bispecific antibodiesthat recognize different epitopes of BCMA or CD3 and BCMA). Otherexemplary compositions comprise more than one BCMA antibody, CD3-BCMAantibody, or BCMA antibody conjugate that recognize the same epitope(s),or different species of BCMA antibodies, CD3-BCMA bispecific antibodies,or BCMA antibody conjugate that bind to different epitopes of BCMA(e.g., human BCMA) or CD3 and BCMA (human CD3 and BCMA).

The composition used in the present invention can further comprisepharmaceutically acceptable carriers, excipients, or stabilizers(Remington: The Science and practice of Pharmacy 21st Ed., 2005,Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations, and may comprise buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrans; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Pharmaceutically acceptable excipients arefurther described herein.

Kits

The invention also provides kits for use in the instant methods. Kits ofthe invention include one or more containers comprising the BCMAantibody, CD3-BCMA bispecific antibody, or the BCMA antibody conjugateas described herein and instructions for use in accordance with any ofthe methods of the invention described herein. Generally, theseinstructions comprise a description of administration of the BCMAantibody, CD3-BCMA bispecific antibody, or the BCMA antibody conjugatefor the above described therapeutic treatments.

The instructions relating to the use of the BCMA antibodies, CD3-BCMAbispecific antibodies, or the BCMA antibody conjugates as describedherein generally include information as to dosage, dosing schedule, androute of administration for the intended treatment. The containers maybe unit doses, bulk packages (e.g., multi-dose packages) or sub-unitdoses. Instructions supplied in the kits of the invention are typicallywritten instructions on a label or package insert (e.g., a paper sheetincluded in the kit), but machine-readable instructions (e.g.,instructions carried on a magnetic or optical storage disk) are alsoacceptable.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a BCMA antibody, CD3-BCMA bispecific antibody, or a BCMAantibody conjugate. The container may further comprise a secondpharmaceutically active agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

EXAMPLES Example 1: Determination of Kinetics and Affinity ofhBCMA/Human IqG Interactions at 25° C. and/or 37° C.

This example determines the kinetics and affinity of various anti-BCMAantibodies at 25° C. and 37° C.

All experiments were performed on a Bio-Rad Proteon XPR36 surfacePlasmon resonance biosensor (Bio-Rad, Hercules, Calif.). An array ofanti-BCMA antibodies was prepared using an amine-coupling method on aBio-Rad GLC Sensor Chip similar to that described in Abdiche, et al.,Anal. Biochem. 411, 139-151 (2011). The analysis temperature for theimmobilization was 25° C. and the running buffer was HBS-T+ (10 mMHEPES, 150 mM NaCl, 0.05% Tween-20, pH 7.4). Channels were activated inthe analyte (horizontal) direction by injecting a mixture of 1 mM ECDand 0.25 mM NHS for 3 minutes at a flow rate of 30 μL/min. IgGs wereimmobilized on the activated spots by injecting them in the ligand(vertical) direction at 20 μg/mL in 10 mM Acetate pH 4.5 buffer for 1.5minutes at 30 μg/mL. The activated surfaces were blocked by injecting 1Methanolamine, pH 8.5 in the analyte direction for 3 minutes at 30μL/min.

The analysis temperature for the hBCMA binding analysis was 37° C. or25° C. in a running buffer of HBS-T+, supplemented with 1 mg/mL BSA. Akinetic titration method was employed for the interaction analysis asdescribed in Abdiche, et al. The hBCMA (human BCMA) analyte was injectedin the analyte direction using a series of injections from low to highconcentration. The concentrations used were 0.08 nM, 0.4 nM, 2 nM, 10 nMand 50 nM (a 5-membered series, with a 5-fold dilution factor and topconcentration of 50 nM). The association time for a given analytedilution was two minutes. Immediately after the 50 nM hBCMA injection,dissociation was monitored for 2 hours. Prior to the hBCMA analyteinjections, buffer was injected 5 times using the same association anddissociation times at the hBCMA analyte cycles to prepare a buffer blanksensorgram for double-referencing purposes (double referencing asdescribed in Myszka, J. Mol. Recognit. 12, 279-284 (1999).

The sensorgrams were double-referenced and fit to a 1:1 Langmuir withmass transport kinetic titration model in BIAevaluation Software version4.1.1 (GE Lifesciences, Piscataway, N.J.). The sensorgrams and fits areshown in FIG. 1, and the kinetics and affinity parameters for variousanti-BCMA antibodies of the present invention are shown in Tables 6A-6C.

TABLE 6A K_(D) Sample k_(a) (1/Ms) k_(d) (1/s) t_(1/2) (min) (pM)A02_Rd4_6nM_C01 1.2E+06 2.8E−05 411 24 A02_Rd4_6nM_C16 1.1E+06 6.2E−05187 59 Combo_Rd4_0.6nM_C29 6.6E+06 1.4E−04 83 21 L3PY/H3TAQ 2.6E+061.4E−04 84 53

TABLE 6B ka (1/Ms) huBCMA @ kd (1/s) huBCMA T½ (min) to huBCMA KD (nM)to huBCMA @ Antibody 25° C. @25° C. @25° C. 25° C. P6E01/P6E01 1.04E+064.15E−03 2.8 4.0 P6E01/H3.AQ 8.35E+05 3.45E−04 33.53 0.41L1.LGF/L3.KW/P6E01 8.31E+05 7.55E−03 1.53 9.08 L1.LGF/L3.NY/P6E011.33E+06 4.40E−03 2.63 3.32 L1.GDF/L3.NY/P6E01 1.60E+06 5.92E−03 1.953.70 L1.LGF/L3.KW/H3.AL 4.28E+05 1.23E−03 9.40 2.87 L1.LGF/L3.KW/H3.AP9.28E+05 2.27E−03 5.10 2.44 L1.LGF/L3.KW/H3.AQ 5.24E+05 9.56E−04 12.091.82 L1.LGF/L3.PY/H3.AP 4.57E+05 9.69E−04 11.92 2.12 L1.LGF/L3.PY/H3.AQ9.31E+05 8.86E−04 13.04 0.95 L1.LGF/L3.NY/H3.AL 7.63E+05 9.70E−04 11.911.27 L1.LGF/L3.NY/H3.AP 9.36E+05 5.33E−04 21.67 0.57 L1.LGF/L3.NY/H3.AQ6.66E+05 2.99E−04 38.61 0.45 L1.GDF/L3.KW/H3.AL 4.45E+05 3.90E−03 2.968.76 L1.GDF/L3.KW/H3.AP 1.17E+06 4.61E−03 2.51 3.93 L1.GDF/L3.KW/H3.AQ7.97E+05 3.48E−03 3.32 4.37 L1.GDF/L3.PY/H3.AQ 1.42E+06 1.35E−02 0.869.49 L1.GDF/L3.NY/H3.AL 9.07E+05 4.03E−03 2.87 4.44 L1.GDF/L3.NY/H3.AP1.41E+06 1.41E−03 8.21 1.00 L1.GDF/L3.NY/H3.AQ 9.84E+05 7.22E−04 16.000.73 L3.KW/P6E01 7.40E+05 3.15E−04 36.66 0.43 L3.PY/P6E01 7.12E+052.28E−04 50.74 0.32 L3.NY/P6E01 8.76E+05 3.84E−04 30.08 0.44 ka (1/Ms)huBCMA @ kd (1/s) huBCMA T½ (min) to huBCMA KD (nM) to huBCMA @ Antibody37° C. @37° C. @37° C. 37° C. L3.PY/L1.PS/P6E01 2.49E+06 1.13E−03 10.210.45 L3.PY/L1.AH/P6E01 2.55E+06 1.26E−03 9.19 0.49 L3.PY/L1.FF/P6E012.39E+06 1.41E−03 8.18 0.59 L3.PY/L1.PH/P6E01 2.81E+06 9.13E−04 12.650.32 L3.PY/L3.KY/P6E01 3.18E+06 1.09E−03 10.65 0.34 L3.PY/L3.KF/P6E012.88E+06 2.08E−03 5.56 0.72 L3.PY/H2.QR 2.56E+06 1.19E−03 9.75 0.46L3.PY/H2.DY 2.60E+06 1.38E−03 8.37 0.53 L3.PY/H2.YQ 2.58E+06 1.56E−037.41 0.60 L3.PY/H2.LT 2.40E+06 1.29E−03 8.95 0.54 L3.PY/H2.HA 2.43E+061.47E−03 7.89 0.60 L3.PY/H2.QL 2.64E+06 2.18E−03 5.31 0.82 L3.PY/H3.YA3.15E+06 1.18E−03 9.82 0.37 L3.PY/H3.AE 3.29E+06 1.39E−03 8.32 0.42L3.PY/H3.AQ 3.08E+06 1.73E−03 6.69 0.56 L3.PY/H3.TAQ 3.08E+06 1.14E−0310.13 0.37 L3.PY/P6E01 2.65E+06 1.96E−03 5.91 0.74 L3.PY/L1.PS/H2.QR3.97E+06 1.03E−01 0.11 25.85 L3.PY/L1.PS/H2.DY 3.22E+06 3.61E−03 3.201.12 L3.PY/L1.PS/H2.YQ 3.35E+06 4.30E−03 2.69 1.28 L3.PY/L1.PS/H2.LT3.40E+06 4.65E−03 2.49 1.37 L3.PY/L1.PS/H2.HA 3.30E+06 1.06E−02 1.093.21 L3.PY/L1.PS/H2.QL 1.52E+07 3.14E−01 0.04 20.64 L3.PY/L1.PS/H3.YA3.07E+06 9.05E−03 1.28 2.95 L3.PY/L1.PS/H3.AE 3.14E+06 1.46E−03 7.930.46 L3.PY/L1.PS/H3.AQ 3.26E+06 1.79E−03 6.46 0.55 L3.PY/L1.PS/H3.TAQ3.25E+06 2.46E−03 4.70 0.76 L3.PY/L1.AH/H2.QR 3.13E+06 1.81E−03 6.390.58 L3.PY/L1.AH/H2.DY 3.05E+06 1.52E−03 7.62 0.50 L3.PY/L1.AH/H2.YQ2.42E+06 1.93E−03 6.00 0.80 L3.PY/L1.AH/H2.LT 3.16E+06 1.23E−03 9.380.39 L3.PY/L1.AH/H2.HA 3.33E+06 1.81E−03 6.37 0.54 L3.PY/L1.AH/H2.QL3.04E+06 1.60E−03 7.22 0.53 L3.PY/L1.AH/H3.YA 3.00E+06 1.50E−03 7.730.50 L3.PY/L1.AH/H3.AE 3.32E+06 1.73E−03 6.70 0.52 L3.PY/L1.AH/H3.AQ3.03E+06 1.97E−03 5.85 0.65 L3.PY/L1.AH/H3.TAQ 3.27E+06 1.19E−03 9.680.37 L3.PY/L1.FF/H2.QR 3.47E+06 1.77E−03 6.54 0.51 L3.PY/L1.FF/H2.DY4.14E+06 2.71E−03 4.27 0.65 L3.PY/L1.FF/H2.YQ 3.32E+06 1.52E−03 7.610.46 L3.PY/L1.FF/H2.LT 3.30E+06 1.67E−03 6.92 0.51 L3.PY/L1.FF/H2.HA3.49E+06 2.19E−03 5.29 0.63 L3.PY/L1.FF/H2.QL 3.48E+06 1.40E−03 8.280.40 L3.PY/L1.FF/H3.YA 3.50E+06 1.80E−03 6.41 0.51 L3.PY/L1.FF/H3.AE3.82E+06 2.63E−03 4.39 0.69 L3.PY/L1.FF/H3.AQ 3.32E+06 1.54E−03 7.510.46 L3.PY/L1.FF/H3.TAQ 3.52E+06 1.89E−03 6.12 0.54 L3.PY/L1.PH/H2.QR3.69E+06 2.36E−03 4.89 0.64 L3.PY/L1.PH/H2.HA 2.37E+06 1.16E−03 9.990.49 L3.PY/L1.PH/H3.AE 3.68E+06 1.34E−03 8.61 0.36 L3.PY/L1.PH/H3.AQ3.08E+06 1.59E−03 7.27 0.52 L3.PY/L1.PH/H3.TAQ 3.58E+06 2.13E−03 5.430.59 L3.PY/L3.KY/H2.QR 2.95E+06 9.90E−04 11.67 0.34 L3.PY/L3.KY/H2.DY3.19E+06 6.42E−04 18.00 0.20 L3.PY/L3.KY/H2.YQ 2.14E+06 1.65E−03 7.020.77 L3.PY/L3.KY/H2.LT 2.92E+06 9.06E−04 12.75 0.31 L3.PY/L3.KY/H2.HA3.29E+06 1.63E−03 7.10 0.49 L3.PY/L3.KY/H2.QL 3.65E+06 2.08E−03 5.560.57 L3.PY/L3.KY/H3.YA 3.30E+06 9.12E−04 12.67 0.28 L3.PY/L3.KY/H3.TAQ2.79E+06 6.49E−04 17.79 0.23 L3.PY/L3.KF/H2.DY 2.74E+06 1.82E−03 6.350.67 L3.PY/L3.KF/H2.YQ 1.96E+06 2.23E−03 5.18 1.14 L3.PY/L3.KF/H2.LT2.75E+06 1.91E−03 6.05 0.69 L3.PY/L3.KF/H2.QL 2.07E+06 1.25E−03 9.260.60 L3.PY/L3.KF/H3.YA 3.12E+06 1.47E−03 7.85 0.47 L3.PY/L3.KF/H3.AE3.07E+06 1.55E−03 7.44 0.51 L3.PY/L3.KF/H3.AQ 3.48E+06 2.27E−03 5.090.65 L3.PY/L3.KF/H3.TAQ 2.82E+06 1.62E−03 7.12 0.58 ka (1/Ms) cyBCMA @kd (1/s) cyBCMA T½ (min) to cyBCMA KD (nM) to cyBCMA @ Antibody 25° C.@25° C. @25° C. 25° C. P6E01/P6E01 7.02E−02 0.16 115.4 P6E01/H3.AQ1.08E+06 7.40E−03 1.6 6.9 L1.LGF/L3.KW/P6E01 4.55E+05 1.95E−02 0.6 42.8L1.LGF/L3.NY/P6E01 9.20E+05 1.05E−02 1.1 11.4 L1.GDF/L3.NY/P6E011.20E+06 7.67E−03 1.5 6.4 L1.LGF/L3.KW/H3.AL 2.90E+05 1.21E−02 1.0 41.8L1.LGF/L3.KW/H3.AP 5.54E+05 1.54E−02 0.7 27.8 L1.LGF/L3.KW/H3.AQ5.27E+05 3.55E−03 3.3 6.7 L1.LGF/L3.PY/H3.AP 3.64E+05 1.30E−02 0.9 35.8L1.LGF/L3.PY/H3.AQ 1.00E+06 4.77E−03 2.4 4.8 L1.LGF/L3.NY/H3.AL 6.35E+051.48E−02 0.8 23.2 L1.LGF/L3.NY/H3.AP 8.30E+05 5.57E−03 2.1 6.7L1.LGF/L3.NY/H3.AQ 7.51E+05 1.48E−03 7.8 2.0 L1.GDF/L3.KW/H3.AL 3.18E+051.80E−02 0.6 56.7 L1.GDF/L3.KW/H3.AP 8.14E+05 2.03E−02 0.6 24.9L1.GDF/L3.KW/H3.AQ 8.02E+05 5.65E−03 2.0 7.0 L1.GDF/L3.PY/H3.AQ 1.55E+061.66E−02 0.7 10.7 L1.GDF/L3.NY/H3.AL 9.00E+05 2.19E−02 0.5 24.3L1.GDF/L3.NY/H3.AP 1.36E+06 7.02E−03 1.6 5.2 L1.GDF/L3.NY/H3.AQ 1.18E+061.36E−03 8.5 1.2 L3.KW/P6E01 7.63E+05 2.57E−03 4.5 3.4 L3.PY/P6E018.55E+05 2.93E−03 3.9 3.4 L3.NY/P6E01 1.01E+06 2.87E−03 4.0 2.8 ka(1/Ms) cyBCMA @ kd (1/s) cyBCMA T½ (min) to cyBCMA KD (nM) to cyBCMA @Antibody 37° C. @37° C. @37° C. 37° C. L3.PY/L1.PS/P6E01 2.17E+066.06E−03 1.91 2.79 L3.PY/L1.AH/P6E01 2.16E+06 5.72E−03 2.02 2.65L3.PY/L1.FF/P6E01 2.45E+06 5.91E−03 1.96 2.41 L3.PY/L1.PH/P6E01 2.17E+067.89E−03 1.46 3.63 L3.PY/L3.KY/P6E01 2.27E+06 5.02E−03 2.30 2.21L3.PY/L3.KF/P6E01 2.39E+06 8.30E−03 1.39 3.48 L3.PY/H2.QR 2.18E+066.58E−03 1.76 3.02 L3.PY/H2.DY 2.24E+06 6.18E−03 1.87 2.76 L3.PY/H2.YQ2.46E+06 6.21E−03 1.86 2.53 L3.PY/H2.LT 2.09E+06 7.57E−03 1.53 3.63L3.PY/H2.HA 1.99E+06 7.55E−03 1.53 3.79 L3.PY/H2.QL 2.05E+06 1.26E−020.91 6.16 L3.PY/H3.YA 2.87E+06 5.40E−03 2.14 1.88 L3.PY/H3.AE 2.82E+065.04E−03 2.29 1.79 L3.PY/H3.AQ 2.77E+06 5.39E−03 2.14 1.94 L3.PY/H3.TAQ2.57E+06 4.37E−03 2.64 1.70 L3.PY/P6E01 2.20E+06 1.31E−02 0.88 5.96L3.PY/L1.PS/H2.QR 5.25E+05 6.70E−04 17.23 1.28 L3.PY/L1.PS/H2.DY1.90E+06 3.78E−03 3.06 1.99 L3.PY/L1.PS/H2.YQ 2.00E+06 3.74E−03 3.091.87 L3.PY/L1.PS/H2.LT 2.17E+06 4.11E−03 2.81 1.89 L3.PY/L1.PS/H2.HA1.45E+06 2.69E−03 4.30 1.86 L3.PY/L1.PS/H2.QL 6.57E+05 6.36E−04 18.170.97 L3.PY/L1.PS/H3.YA 1.77E+06 9.98E−03 1.16 5.65 L3.PY/L1.PS/H3.AE2.46E+06 4.13E−03 2.80 1.68 L3.PY/L1.PS/H3.AQ 2.52E+06 4.33E−03 2.671.72 L3.PY/L1.PS/H3.TAQ 2.58E+06 5.52E−03 2.09 2.14 L3.PY/L1.AH/H2.QR2.20E+06 4.91E−03 2.35 2.23 L3.PY/L1.AH/H2.DY 2.32E+06 4.51E−03 2.561.95 L3.PY/L1.AH/H2.YQ 1.58E+06 4.31E−03 2.68 2.74 L3.PY/L1.AH/H2.LT2.19E+06 2.96E−03 3.91 1.35 L3.PY/L1.AH/H2.HA 2.58E+06 4.39E−03 2.631.70 L3.PY/L1.AH/H2.QL 2.62E+06 9.55E−03 1.21 3.65 L3.PY/L1.AH/H3.YA2.37E+06 5.26E−03 2.20 2.22 L3.PY/L1.AH/H3.AE 2.25E+06 3.56E−03 3.251.58 L3.PY/L1.AH/H3.AQ 2.24E+06 3.99E−03 2.90 1.78 L3.PY/L1.AH/H3.TAQ2.28E+06 3.02E−03 3.83 1.32 L3.PY/L1.FF/H2.QR 2.55E+06 4.21E−03 2.751.65 L3.PY/L1.FF/H2.DY 2.66E+06 5.00E−03 2.31 1.88 L3.PY/L1.FF/H2.YQ2.19E+06 3.26E−03 3.55 1.49 L3.PY/L1.FF/H2.LT 2.19E+06 3.41E−03 3.381.56 L3.PY/L1.FF/H2.HA 2.33E+06 4.17E−03 2.77 1.79 L3.PY/L1.FF/H2.QL2.36E+06 4.49E−03 2.57 1.91 L3.PY/L1.FF/H3.YA 2.46E+06 4.16E−03 2.771.69 L3.PY/L1.FF/H3.AE 2.85E+06 5.01E−03 2.31 1.76 L3.PY/L1.FF/H3.AQ2.18E+06 3.29E−03 3.51 1.51 L3.PY/L1.FF/H3.TAQ 2.32E+06 3.76E−03 3.071.62 L3.PY/L1.PH/H2.QR 2.42E+06 4.36E−03 2.65 1.80 L3.PY/L1.PH/H2.HA1.61E+06 5.53E−03 2.09 3.44 L3.PY/L1.PH/H3.AE 2.61E+06 2.02E−03 5.720.77 L3.PY/L1.PH/H3.AQ 2.28E+06 3.41E−03 3.39 1.50 L3.PY/L1.PH/H3.TAQ2.51E+06 3.20E−03 3.61 1.28 L3.PY/L3.KY/H2.QR 2.05E+06 7.74E−03 1.493.78 L3.PY/L3.KY/H2.DY 1.96E+06 2.43E−03 4.75 1.24 L3.PY/L3.KY/H2.YQ1.27E+06 2.58E−03 4.47 2.04 L3.PY/L3.KY/H2.LT 1.82E+06 2.32E−03 4.981.27 L3.PY/L3.KY/H2.HA 2.28E+06 3.18E−03 3.63 1.40 L3.PY/L3.KY/H2.QL2.75E+06 4.09E−03 2.83 1.49 L3.PY/L3.KY/H3.YA 1.84E+06 4.28E−03 2.702.33 L3.PY/L3.KY/H3.TAQ 1.81E+06 1.92E−03 6.03 1.06 L3.PY/L3.KF/H2.DY2.08E+06 3.68E−03 3.14 1.77 L3.PY/L3.KF/H2.YQ 1.41E+06 5.01E−03 2.303.55 L3.PY/L3.KF/H2.LT 1.91E+06 4.13E−03 2.80 2.16 L3.PY/L3.KF/H2.QL1.42E+06 3.10E−03 3.73 2.18 L3.PY/L3.KF/H3.YA 2.10E+06 7.96E−03 1.453.78 L3.PY/L3.KF/H3.AE 1.85E+06 5.64E−03 2.05 3.05 L3.PY/L3.KF/H3.AQ2.55E+06 2.38E−03 4.85 0.93 L3.PY/L3.KF/H3.TAQ 2.01E+06 1.91E−03 6.050.95

TABLE 6C* Human Cyno Antibody ka (1/Ms) kd (1/s) KD (pM) ka (1/Ms) kd(1/s) KD (pM) P5A2_VHVL 6.96E+06 3.87E−02 5567 1.61E+06 1.64E−02 10230A02_Rd4_0.6 nM_C06 3.49E+06 7.37E−05 21 1.81E+06 1.05E−04 58 A02_Rd4_0.6nM_C09 5.50E+06 9.75E−05 18 2.13E+06 1.74E−04 82 A02_Rd4_6 nM_C161.56E+06 1.41E−04 90 1.34E+06 1.58E−04 118 A02_Rd4_6 nM_C03 1.69E+061.26E−04 75 1.17E+06 1.85E−04 158 A02_Rd4_6 nM_C01 3.11E+06 9.20E−05 301.45E+06 5.83E−04 401 A02_Rd4_6 nM_C26 4.26E+06 1.39E−04 33 2.21E+064.48E−04 203 A02_Rd4_6 nM_C25 2.75E+06 1.80E−04 65 1.50E+06 3.30E−04 220A02_Rd4_6 nM_C22 3.38E+06 1.82E−04 54 1.84E+06 3.24E−04 176 A02_Rd4_6nM_C19 3.00E+06 1.48E−04 49 2.54E+06 6.61E−04 260 A02_Rd4_0.6 nM_C034.27E+06 1.82E−04 43 2.12E+06 4.26E−04 201 A02_Rd4_6 nM_C07 1.48E+061.89E−04 128 6.91E+05 7.86E−04 1138 A02_Rd4_6 nM_C23 1.22E+07 2.55E−0421 2.63E+06 4.14E−04 157 A02_Rd4_0.6 nM_C18 4.73E+06 2.29E−04 483.24E+06 6.39E−04 197 A02_Rd4_6 nM_C10 4.51E+06 3.15E−04 70 1.90E+068.98E−04 472 A02_Rd4_6 nM_C05 3.10E+06 3.08E−04 99 1.36E+06 1.29E−03 950A02_Rd4_0.6 nM_C10 2.30E+06 2.96E−04 129 8.83E+05 1.63E−03 1842A02_Rd4_6 nM_C04 4.47E+06 6.03E−04 135 2.18E+06 8.31E−04 381 A02_Rd4_0.6nM_C26 7.26E+06 4.43E−04 61 2.71E+06 2.56E−03 941 A02_Rd4_0.6 nM_C138.53E+06 5.66E−04 66 2.29E+06 1.28E−03 560 A02_Rd4_0.6 nM_C01 4.74E+069.15E−04 193 2.39E+06 1.57E−03 655 A02_Rd4_6 nM_C08 3.92E+06 7.38E−04188 2.23E+06 1.13E−02 5072 P5C1_VHVL 1.16E+07 6.92E−02 5986 3.53E+065.38E−02 15231 C01_Rd4_6 nM_C24 7.47E+06 3.48E−03 467 3.17E+06 8.91E−04281 C01_Rd4_6 nM_C26 1.50E+07 1.36E−03 90 4.75E+06 1.99E−03 419C01_Rd4_6 nM_C02 1.61E+07 1.44E−03 89 5.12E+06 2.18E−03 426 C01_Rd4_6nM_C10 1.31E+07 2.12E−03 162 4.44E+06 2.19E−03 493 C01_Rd4_0.6 nM_C271.23E+07 3.74E−03 303 3.34E+06 2.85E−03 852 C01_Rd4_6 nM_C20 6.02E+062.76E−03 459 3.60E+06 6.25E−03 1737 C01_Rd4_6 nM_C12 1.21E+07 6.49E−03535 4.51E+06 3.70E−03 820 C01_Rd4_0.6 nM_C16 1.55E+07 6.30E−03 4074.95E+06 4.64E−03 939 C01_Rd4_0.6 nM_C09 1.51E+07 8.25E−03 545 5.28E+069.36E−03 1773 C01_Rd4_6 nM_C09 1.58E+07 1.28E−02 811 3.73E+06 8.68E−032328 C01_Rd4_0.6 nM_C03 1.55E+07 1.50E−02 964 4.72E+06 1.19E−02 2528C01_Rd4_0.6 nM_C06 1.82E+07 1.54E−02 847 6.22E+06 1.21E−02 1948C01_Rd4_6 nM_C04 2.33E+07 4.97E−02 2134 6.34E+06 3.27E−02 5156COMBO_Rd4_0.6 nM_C22 1.97E+06 7.15E−05 36 1.34E+06 6.66E−05 50COMBO_Rd4_6 nM_C21 1.17E+07 7.34E−05 6 3.17E+06 2.48E−04 78 COMBO_Rd4_6nM_C10 5.47E+06 9.72E−05 18 1.52E+06 1.60E−04 105 COMBO_Rd4_0.6 nM_C041.07E+07 1.58E−04 15 3.52E+06 1.37E−04 39 COMBO_Rd4_6 nM_C25 7.98E+061.13E−04 14 2.85E+06 2.26E−04 79 COMBO_Rd4_0.6 nM_C21 1.34E+07 1.15E−049 3.63E+06 3.04E−04 84 COMBO_Rd4_6 nM_C11 6.74E+06 1.24E−04 18 2.64E+064.12E−04 156 COMBO_Rd4_0.6 nM_C20 7.65E+06 1.46E−04 19 3.09E+06 2.84E−0492 COMBO_Rd4_6 nM_C09 8.85E+06 1.43E−04 16 2.37E+06 3.18E−04 134COMBO_Rd4_6 nM_C08 8.99E+06 1.69E−04 19 3.06E+06 4.28E−04 140COMBO_Rd4_0.6 nM_C19 7.86E+06 1.55E−04 20 2.92E+06 9.79E−04 336COMBO_Rd4_0.6 nM_C02 8.57E+06 1.85E−04 22 3.01E+06 4.94E−04 164COMBO_Rd4_0.6 nM_C23 7.39E+06 2.10E−04 28 2.81E+06 5.31E−04 189COMBO_Rd4_0.6 nM_C29 1.47E+07 2.77E−04 19 4.00E+06 3.36E−04 84COMBO_Rd4_0.6 nM_C09 1.04E+07 3.19E−04 31 3.77E+06 3.46E−04 92COMBO_Rd4_6 nM_C12 1.38E+07 2.70E−04 20 3.29E+06 4.86E−04 148COMBO_Rd4_0.6 nM_C30 4.35E+06 2.82E−04 65 1.68E+06 8.08E−04 481COMBO_Rd4_0.6 nM_C14 8.66E+06 3.28E−04 38 3.48E+06 6.45E−04 185COMBO_Rd4_6 nM_C07 1.05E+07 3.71E−04 35 3.94E+06 9.34E−04 237COMBO_Rd4_6 nM_C02 1.05E+06 4.43E−04 422 7.95E+05 1.36E−03 1714COMBO_Rd4_0.6 nM_C05 4.32E+06 4.97E−04 115 1.94E+06 1.72E−03 886COMBO_Rd4_0.6 nM_C17 8.68E+06 8.01E−04 92 3.06E+06 1.01E−03 330COMBO_Rd4_6 nM_C22 3.03E+06 7.75E−04 256 1.70E+06 1.65E−03 972COMBO_Rd4_0.6 nM_C11 5.11E+06 1.06E−03 207 2.20E+06 4.23E−03 1924 *Thebinding analysis was conducted at 37° C.

Example 2: Flow Cytometry of Human Anti-BCMA Antibodies on BCMA PositiveTumor Cells

This example demonstrates binding of BCMA positive tumor cells byvarious BCMA antibodies of the present invention.

Binding of human anti-hBCMA expressed in mouse IgG2a were assessed onBCMA-expressing cells (KMS12BM, L363, MM1S and KMS12PE) by flowcytometry. 250,000 cells were incubated with 0.5 ug antibody in 100 uLbinding buffer (PBS (Phosphate Buffered Saline)+0.2% BSA (Bovine SerumAlbumin)), followed by incubation with Alex Fluor 647 conjugatedanti-mouse IgG (Biolegend). Table 7 shows MFI (mean fluorescenceintensity) on BCMA positive tumor cells by various BCMA antibodies(e.g., Combo_Rd4_0.6 nM_C29, A02_Rd4_6 nM_C01, A02_Rd4_6 nM_C16, andP6E01/H3TAQ)

TABLE 7 P6E01/ secondary only Combo_Rd4_0.6nM_C29 A02_Rd4_6nM_C01A02_Rd4_6nM H3TAQ Cell Line MFI KMS12PE (BCMA+++) 26 6114 5862 3094 6018MM1S (BCMA++) 22 2569 2539 1951 2715 L363 (BCMA+) 22 1667 1176 789 1457KMS12BM (BCMA+) 22 583 580 421 634

Example 3: Cytotoxicity of Anti-BCMA ADCs in BCMA Positive Cells

This example illustrates the efficacy of the anti-BCMA ADCs in BCMApositive cells.

Human anti-BCMA (L3.PY/P6E01, L3.PY/H3.TAQ, Combo_Rd4_0.6 nM_C29,A02_Rd4_6 nM_C01, and A02_Rd4_6 nM_C16) antibodies were expressed ashuman IgG1 subtypes engineered with glutamine-containingtransglutaminase (“Q”) tags (e.g. LCQ05, H7c, N297A, N297Q, N297A/H7c,N297Q/LCQ05) for drug antibody ratios (DAR) of 2, 4, and 6. TG17corresponds to SEQ ID NO: 472 (LLQGPP); LCQ05 correspond to SEQ ID NO:474 (GGLLQGPP), H7c correspond to SEQ ID NO: 454 (LLQG), respectively,and conjugated with AcLys-Val-Cit-PABC-Aur0101(Acetyl-Lysine-Valine-Citrulline-p-aminobenzyloxycarbonyl),amino-PEG6-C2-Aur3377, or amino-PEG6-C2-Aur0131 as indicated in Table 8.In one instance, the transglutaminase tags can be engineered at thelight chain, heavy chain, or a combination of light and heavy chains. Inother instance, the transglutaminase tag (e.g., Q) is engineered at siteof the antibody, such as at position 297 of the human IgG (EU numberingscheme). For example, the wild-type amino acid asparagine (N) issubstituted with glutamine or alanine at position 297 of the BCMAantibody (N297Q or N297A) of the present invention. Anti-BCMA antibodyconjugation to Aur0101, Aur3377, and Aur0131 was then achieved viamicrobial transglutaminase-catalyzed transamidation reaction between theanti-BCMA antibody carrying a targeted glutamine or glutamine tag at thespecific site (e.g., carboxyl terminus or amino terminus of the heavychain or light chain, position 297, or at another site of the antibody)and an amine-containing derivative of the payload (e.g., MMAD, Aur0101,Aur3377, or Aur0131). In some instances, the wild-type amino acid lysineat position 222, 340, or 370 (in accordance with EU numbering scheme)was replaced with amino acid arginine (“K222R”, “K340R”, or “K370R”).For example, the K222R substitution was found to have the surprisingeffect of resulting in more homogenous antibody and payload conjugate,better intermolecular crosslinking between the antibody and the payload,and/or significant decrease in interchain crosslinking with theglutamine tag on the C-terminus of the antibody light chain.

In the transamidation reaction, the glutamine on the antibody acted asan acyl donor, and the amine-containing compound acted as an acylacceptor (amine donor). Purified anti-BCMA antibody in the concentrationof 1-150 μM was incubated with a 5-100 molar excess acyl acceptor,ranging between 5 μM-15 mM, in the presence of 0.23-0.55% (w/v)Streptoverticillium mobaraense transglutaminase (ACTIVA™, Ajinomoto,Japan) in 10-1000 mM NaCl, and 25 mM MES, HEPES[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] or Tris HCl bufferat pH range 6.2-8.8. The reaction conditions were adjusted forindividual acyl acceptor derivatives, and the optimal efficiency andspecificity were typically observed for 33 μM antibody, 0.67 mMderivative, and 0.378% (w/v) transglutaminase in 75 mM NaCl, 25 mM TrisHCl, pH 8.5. Following incubation at 20-37 deg C. for 1-24 hours, theantibody was purified on Butyl Sepharose High Performance (Butyl HP)resin (GE Healthcare, Waukesha, Wis.) using standard chromatographymethods known to persons skilled in the art, such as commercialhydrophobic interaction chromatography from GE Healthcare.

Target expressing (MM1.S, KMS12BM and L363) cells were then seeded onclear bottom plates at 3000 cells/well. Cells were treated with 4-foldserially diluted antibody-drug conjugates in triplicates. Cell viabilitywas determined by CellTiter-Glo® Luminescent Cell Viability Assay 96(Promega, Madison Wis.) 96 hours after treatment. Relative cellviability was determined as percentage of untreated control. EC50 wascalculated by Prism software. Table 8 shows that all human anti-BCMAantibodies of the present invention conjugated to cytotoxic agent 0101,3377, and 0131 through transglutaminase tags and linkers exert potentcell killing activity in BCMA expressing cells.

TABLE 8 EC50 MM1.S LUCGFP KMS12BM LUCGFP L363 LUCGFP (BCMA++) (+) (+)ANTIBODY DAR nM ug/mL nM ug/mL nM ug/mL L3.PY/P6E01 4 0.42 0.06 31.794.77 7.34 1.10 N297Q/K222R AcLys vc0101 L3PY/H3.TAQ 4 0.12 0.02 3.940.59 0.60 0.09 N297Q/K222R Aclys vc0101 A02_Rd4_6 nM_C01 3.91 0.26 0.0418.67 2.80 2.53 0.38 N297Q/K222R Aclys vc0101 A02_Rd4_6 nM_C16 3.92 0.800.12 38.73 5.81 9.68 1.45 N297Q/K222R Aclys vc0101 Combo_Rd4_0.6 nM_C292 0.13 0.02 10.91 1.64 1.67 0.25 LCQ05/K222R Aclys vc0101 Combo_Rd4_0.6nM_C29 3.9 0.11 0.02 0.99 0.15 0.66 0.10 N297Q/K222R Aclys vc0101Combo_Rd4_0.6 nM_C29 5.98 0.34 0.05 1.19 0.18 1.05 0.16LCQ05/N297Q/K222R Aclys vc0101 Combo_Rd4_0.6 nM_C29 3.81 0.23 0.03 0.850.13 0.81 0.12 H7c/N297A/K222R AmPEG6 Aur0131 Combo_Rd4_0.6 nM_C29 3.880.30 0.05 5.01 0.75 1.36 0.20 H7c/N297A/K222R AmPEG6 Aur3377

Example 4: Anti-BCMA ADCs Induce Tumor Regression in an OrthotopicMultiple Myeloma Model

This example illustrates the in vivo efficacy of the anti-BCMA ADCs inthe MM1S orthotopic multiple myeloma model.

In vivo efficacy study of BCMA ADCs was performed with multiple myelomacell line MM1.S expressing luciferase and GFP (Green FluorescentProtein) in an orthotopic model. Ten million MM1.S LucGFP cells wereinjected intravenously through the tail vein into 6-8 weeks old femaleCB17/SCID animals. Intraperitoneal injection of D-luciferin (RegisTechnologies, Morton Grove, Ill.) (200 uL per animal at 15 mg/mL),followed by anesthesia with isofluorane and subsequent whole bodybioluminescence imaging (BLI) enable monitoring of tumor burden.Bioluminescent signals emitted by the interaction between luciferaseexpressed by the tumor cells and luciferin were captured by imagingusing an IVIS Spectrum CT (Perkin Elmer, MA) and quantified as totalflux (photons/sec) using Living Image 4.4 (Caliper Life Sciences,Alameda, Calif.). When the total flux reached an average of 1-3E6 forall animals, the animals were randomized into groups and a single doseof a human anti-BCMA antibody conjugated with 1) LCQ05/K222R-vc0101 atthe C-terminus of the antibody light chain and control conjugates wereadministered through bolus tail vein injection. Animals were terminatedwhen they exhibit hindlimb paralysis, an endpoint for MM1.S orthotopicmodels. FIG. 2 shows that a single dose at 3 mg/kg of various humananti-BCMA ADCs inhibits tumor progression as compared to the negativecontrol (NNC), including P6E01/P6E01-AcLys-Val-Cit-PABC-Aur0101;P5A2_VHVL-AcLys-Val-Cit-PABC-Aur0101;P5C1_VHVL-AcLys-Val-Cit-PABC-Aur0101; P4G4-AcLys-Val-Cit-PABC-Aur0101;and P1A11-AcLys-Val-Cit-PABC-Aur0101.

This study demonstrates that treatment with a BCMA-ADC inhibitsprogression of multiple myeloma.

Example 5: Anti-BCMA ADCs Induce Tumor Regression and Inhibition in anOrthotopic Multiple Myeloma Model

This example also illustrates the in vivo efficacy of the anti-BCMA ADCsin the MM1.S orthotopic multiple myeloma models.

In vivo efficacy study of BCMA ADCs was performed with multiple myelomacell line MM1.S expressing luciferase and GFP in an orthotopic model.Ten million MM1.S LucGFP cells were injected intravenously through thetail vein into 6-8 weeks old female CB17/SCID animals. Intraperitonealinjection of D-luciferin (Regis Technologies, Morton Grove, Ill.) (200uL per animal at 15 mg/mL), followed by anesthesia with isofluorane andsubsequent whole body bioluminescence imaging (BLI) enable monitoring oftumor burden. Bioluminescent signals emitted by the interaction betweenluciferase expressed by the tumor cells and luciferin were captured byimaging using an IVIS Spectrum CT (Perkin Elmer, MA) and quantified astotal flux (photons/sec) using Living Image 4.4 (Caliper Life Sciences,Alameda, Calif.). When the total flux reached an average of 1-3E6 forall animals, the animals were randomized into groups; 1)H7c/N297A/K222R-amino-PEG6-C2-3377, 2)N297Q/K222R-AcLys-Val-Cit-PABC-Aur0101, 3)LCQ05/K222R-AcLys-Val-Cit-PABC-Aur0101, 4)H7c/N297A/K222R-amino-PEG6-C2-0131, 5)N297Q/K222R/LCQ05-AcLys-Val-Cit-PABC-Aur0101, and 6) control conjugateLCQ04/K222R-AcLys-Val-Cit-PABC-Aur0101. A single dose of human anti-BCMAADCs and control conjugate were administered through bolus tail veininjection. Animals were terminated when they exhibit hindlimb paralysis,an endpoint for MM1.S orthotopic model. FIG. 3 shows that a single doseof human anti-BCMA L3.PY/P6E01 antibody conjugated with 1)H7c/N297A/K222R-amino-PEG6-C2-0131 and 2)H7c/N297A/K222R-amino-PEG6-C2-3377 resulted in tumor regression. Asingle dose of human anti-BCMA L3.PY/P6E01 antibody conjugated with 1)N297Q/K222R-AcLys-Val-Cit-PABC-Aur0101, 2)LCQ05/K222R-AcLys-Val-Cit-PABC-Aur0101, and 3)N297Q/K222R/LCQ05-AcLys-Val-Cit-PABC-Aur0101 resulted in tumorinhibition.

Accordingly, this study demonstrates that treatment with a BCMA-ADCinduces regression and inhibits progression of multiple myeloma.

Example 6: Anti-BCMA ADCs Induce Tumor Inhibition in an OrthotopicMultiple Myeloma Model

This example also illustrates the in vivo efficacy of the anti-BCMA ADCsin the KMS12BM orthotopic multiple myeloma models

In vivo efficacy study of BCMA ADCs was performed with multiple myelomacell line KMS12BM expressing luciferase and GFP in an orthotopic model.6-8 weeks old female NSG animals were irradiated with 100 cGy and 24hours post irradiation, ten million KMS12BM LucGFP cells were injectedintravenously through the tail vein. Intraperitoneal injection ofD-luciferin (Regis Technologies, Morton Grove, Ill.) (200 uL per animalat 15 mg/mL), followed by anesthesia with isofluorane and subsequentwhole body bioluminescence imaging (BLI) enable monitoring of tumorburden. Bioluminescent signals emitted by the interaction betweenluciferase expressed by the tumor cells and luciferin are captured byimaging using an IVIS Spectrum CT (Perkin Elmer, MA) and quantified astotal flux (photons/sec) using Living Image 4.4 (Caliper Life Sciences,Alameda, Calif.). When the total flux reached an average of 5E6 for allanimals, the animals were randomized into groups; 1)H7c/N297A/K222R-amino-PEG6-C2-3377, 2)N297Q/K222R-AcLys-Val-Cit-PABC-Aur010, 3)LCQ05/K222R-AcLys-Val-Cit-PABC-Aur0101, 4)H7c/N297A/K222R-amino-PEG6-C2-0131, 5)N297Q/K222R/LCQ05-AcLys-Val-Cit-PABC-Aur0101, and 6) control conjugateLCQ04/K222R-AcLys-Val-Cit-PABC-Aur0101. A single dose of human anti-BCMAADCs and control conjugate was administered through bolus tail veininjection. Animals were terminated when they lose more than 15% of totalbody weight, an endpoint for KMS12BM orthotopic models. FIG. 4 showsthat a single dose of human anti-BCMA L3.PY/P6E01 antibody conjugatedwith 1) H7c/N297A/K222R-amino-PEG6-C2-3377, 2)N297Q/K222R-AcLys-Val-Cit-PABC-Aur0101, 3)LCQ05/K222R-AcLys-Val-Cit-PABC-Aur0101, 4)H7c/N297A/K222R-amino-PEG6-C2-0131, and 5)N297Q/K222R/LCQ05-AcLys-Val-Cit-PABC-Aur0101 resulted in tumorinhibition.

Accordingly, this study further demonstrates that treatment with aBCMA-ADC induces regression and inhibits progression of multiplemyeloma.

Example 7: Dose Response Curve of Anti-BCMA ADC in MM1S Orthotopic Model

This example further illustrates the in vivo efficacy of the anti-BCMAADCs in the MM1S orthotopic multiple myeloma models

In vivo efficacy study of BCMA ADCs was performed with multiple myelomacell line MM1.S expressing luciferase and GFP in an orthotopic model.Ten million MM1.S LucGFP cells were injected intravenously through thetail vein into 6-8 weeks old female CB17/SCID animals. Intraperitonealinjection of D-luciferin (Regis Technologies, Morton Grove, Ill.) (200uL per animal at 15 mg/mL), followed by anesthesia with isofluorane andsubsequent whole body bioluminescence imaging (BLI) enable monitoring oftumor burden. Bioluminescent signals emitted by the interaction betweenluciferase expressed by the tumor cells and luciferin were captured byimaging using an IVIS Spectrum CT (Perkin Elmer, MA) and quantified astotal flux (photons/sec) using Living Image 4.4 (Caliper Life Sciences,Alameda, Calif.). When the total flux reached an average of 1.2E6 forall animals, the animals were randomized into groups; 1) 0.1 mg/kgH7c/N297A/K222R-amino-PEG6-C2-0131, 2) 0.38 mg/kgH7c/N297A/K222R-amino-PEG6-C2-0131, 3) 0.75 mg/kgH7c/N297A/K222-amino-PEG6-C2-0131, 4) 1.5 mg/kgH7c/N297A/K222R-amino-PEG6-C2-0131, and 5) 3 mg/kg control conjugateN297Q/K222R-AcLys-VC-0101. A single dose of human anti-BCMA ADCs andcontrol conjugate were administered through bolus tail vein injection.Animals were terminated when they exhibit hindlimb paralysis, anendpoint for MM1.S orthotopic model. FIG. 5 shows that a single dose ofhuman anti-BCMA COMBO_Rd4_0.6 nM_C29 antibody conjugated with groups1)-4) above resulted in tumor regression starting at 0.1 mg/kg and tumorinhibition up to 100 days starting at 0.75 mg/kg.

Accordingly, this study demonstrates that treatment with a BCMA-ADCinduces tumor regression and tumor inhibition in multiple myeloma.

Example 8: Generation and Purification of Heterodimeric Antibodies

This example describes the generation and purification of theheterodimeric antibodies of the present application.

The variable region of the human specific anti-CD3 antibody was clonedinto a human IgG1 or IgG2ΔA containing the following mutations 221R,228R, and K409R; or 223R, 225R, 228R, and K409R, respectively, andreferred as hIgG1 RRR or IgG2ΔA-RRRR.

The variable region of the anti-target antibody was cloned into a humanIgG1 or IgG2ΔA containing the following mutations 221E, 228E, L368E or223E, 225E, 228E and L368E, respectively, and referred as hIgG1EEE orhIgG2ΔA-EEEE.

Heterodimers were prepared by incubation of the anti-CD3 IgG1 or IgG2ΔAhaving hIgG1 RRR or IgG2ΔA-RRRR mutations with an anti-target antibodyhaving hIgG1 EEE or hIgG2ΔA-EEEE mutations in PBS with 1 mM or 2 mM GSHfor 24 hrs at 37° C. as described in International Patent ApplicationNo. PCT/US2011/036419 (WO2011/143545). The heterodimer was purified byion exchange chromatography, as described below.

All the heterodimers were purified by ion exchange chromatography.Briefly, analytical ion exchange separation of the Fc-hetero andFc-homodimers was carried out on Agilent 1100 quaternary pump LC system(Agilent Inc, Santa Clara, Calif., USA) equipped with weak cationexchange DIONEX Propac WCX-10G (4×50 mm) column. Proteins were injectedin 5% buffer A (20 mM MES pH 5.4) and eluted in a gradient from 25% to75% buffer B (20 mM MES pH 5.4 and 500 mM NaCl) over a 20 minute periodwith 1 ml/min flow rate. Larger scale Fc-heterodimer purification wasperformed on an Akta Explorer (GE) equipped with weak cation exchangeDIONEX Propac WCX-10G (4×250 mm) column. Proteins were injected in 5%buffer A (20 mM MES pH 5.4) and eluted in a gradient from 15% to 75%buffer B (20 mM MES pH 5.4 and 500 mM NaCl) over a 60 minute period with1 ml/min flow rate.

Example 9: Determination of Kinetics and Affinity of hCD3/Human IgGInteractions at 25° C. and/or 37° C.

This example determines the kinetics and affinity of various anti-CD3antibodies at 25° C. and 37° C.

All experiments were performed on a Bio-Rad Proteon XPR36 surfacePlasmon resonance biosensor (Bio-Rad, Hercules, Calif.). An array ofanti-CD3 antibodies was prepared using an amine-coupling method on aBio-Rad GLC Sensor Chip similar to that described in Abdiche, et al.,Anal. Biochem. 411, 139-151 (2011). The analysis temperature for theimmobilization was 25° C. and the running buffer was HBS-T+(10 mM HEPES,150 mM NaCl, 0.05% Tween-20, pH 7.4). Channels were activated in theanalyte (horizontal) direction by injecting a mixture of 1 mM ECD and0.25 mM NHS for 3 minutes at a flow rate of 30 μL/min. IgGs wereimmobilized on the activated spots by injecting them in the ligand(vertical) direction at 20 μg/mL in 10 mM Acetate pH 4.5 buffer for 1.5minutes at 30 μg/mL. The activated surfaces were blocked by injecting 1Methanolamine, pH 8.5 in the analyte direction for 3 minutes at 30μL/min.

The analysis temperature for the hCD3 binding analysis was 37° C. or 25°C. in a running buffer of HBS-T+, supplemented with 1 mg/mL BSA. Akinetic titration method was employed for the interaction analysis asdescribed in Abdiche, et al. The hCD3 (human CD3) analyte was injectedin the analyte direction using a series of injections from low to highconcentration. The concentrations used were 0.08 nM, 0.4 nM, 2 nM, 10 nMand 50 nM (a 5-membered series, with a 5-fold dilution factor and topconcentration of 50 nM). The association time for a given analytedilution was two minutes. Immediately after the 50 nM hCD3 injection,dissociation was monitored for 2 hours. Prior to the hCD3 analyteinjections, buffer was injected 5 times using the same association anddissociation times at the hCD3 analyte cycles to prepare a buffer blanksensorgram for double-referencing purposes (double referencing asdescribed in Myszka, J. Mol. Recognit. 12, 279-284 (1999).

The sensorgrams were double-referenced and fit to a 1:1 Langmuir withmass transport kinetic titration model in BIAevaluation Software version4.1.1 (GE Lifesciences, Piscataway, N.J.). The kinetics and affinityparameters for various anti-CD3 antibodies of the present invention areshown in Table 9.

TABLE 9 ka (1/Ms) huCD3ed kd (1/s) huCD3ed T½ (min) to huCD3ed KD (nM)to huCD3ed Antibody @25° C. @25° C. @25° C. @25° C. H2B4 3.7E+05 2.0E−035.8 5.3 ka (1/Ms) huCD3ed kd (1/s) huCD3ed T½ (min) to huCD3ed KD (nM)to huCD3ed Antibody @37° C. @37° C. @37° C. @37° C. H2B4 4.37E+050.01369 0.84 3.14E−08 h2B4-VH-wt VL_TK 3.80E+05 1.40E−02 0.83 3.80E−08h2B4-VH-Hnps VL_TK 3.90E+05 1.60E−02 0.72 4.10E−08 h2B4-VH-yads VL_TK2.40E+05 2.10E−02 0.79 8.60E−08 h2B4-VH-yaes VL_TK 2.30E+05 2.30E−020.50 1.00E−07 h2B4-VH-yaps VL_TK 2.50E+05 2.30E−02 0.50 9.20E−08 ka(1/Ms) cyCD3ed @ kd (1/s) cyCD3ed T½ (min) to cyCD3ed KD (nM) to cyCD3ed@ Antibody 25° C. @25° C. @25° C. 25° C. H2B4 3.9E+05 1.5E−03 7.7 3.8

Example 10: Flow Cytometry of Human Anti-CD3 Bispecific Antibodies on BCells and CD8+ T Cells

This example demonstrates the efficacy of the anti-CD3-anti-CD20bispecific antibodies in CD20+ cells.

Cynomolgus monkey studies were conducted at Charles River Laboratories,Preclinical Services Nevada in accordance with the Institutional AnimalCare and Use Committee. Animals (n=2) were dosed via intravenous bolusinjection with the bispecific anti-CD20/h2B4 antibody at doses of 500ug/kg, 100 ug/kg, 20 ug/kg, 2 ug/kg, 0.2 ug/kg or 0.02 ug/kg. Animalswere observed twice daily and at each blood collection time point. Bloodfor flow cytometry and cytokine analysis was collected into K2EDTA tubesfrom a peripheral vessel not used for i.v. dosing.

Efficacy was determined by measuring B cells and T cells in peripheralblood by flow cytometry. Whole blood was collected at the time pointsindicated and kept at 4° C. until analysis. Erythrocytes were lysed withACK buffer (Gibco) for 5 minutes at room temperature and white bloodcells were pelleted by centrifugation. Cells were stained for 1 hr. at4° C. with a cocktail containing fluorescently labeled antibodiesrecognizing cyno CD19 (Beckman Coulter), CD45, CD4, CD8, Ki67 (BDBiosciences) in PBS+2% FBS. For Ki67 analysis, cells were first stainedwith CD4 and CD8, then fix/permeabilized with BD cyotfix/cytoperm kit(BD Biosciences) according to manufacturer's instructions prior tointracellular staining for Ki67. Acquisition of cells on a BD LSRII flowcytometer was carried out immediately after staining.

The resulting B cell count was graphed as a percentage of the pre-studyB cell count in FIGS. 6A-6F. Prolonged B cell depletion following asingle dose was achieved with doses as low as 2 ug/kg. B cell depletionwas seen at all doses. The duration of the depletion effect was dosedependent.

The resulting CD8+ T cell count was graphed as percentage of thepre-study CD8+ T cell count in FIGS. 7A-7F. After an initialrelocalization, T cell levels were restored to baseline levels or abovefor the duration of the study.

Example 11: Flow Cytometry of Human Anti-CD3 Bispecific Antibodies onCD8+ T Cells

This example demonstrates the efficacy of the monovalent anti-CD3antibody on T cell kinetics and activation.

Cynomolgus monkey studies were conducted and efficacy was determined bymeasuring T cells in peripheral blood by flow cytometry as described inExample 3. Cynomolgus monkeys (n=2) were dosed weekly, i.v., at 0.2ug/kg with anti-CD20/h2B4 or NNC (non-specific antibody)/h2B4. Incontrast to the CD20 targeted bispecific antibody, the NNC/h2B4 haslittle to no effect on CD8+ T cell kinetics in the blood as measured byflow cytometry. Ki67 was used as a marker for T cell activation.

The resulting T cell count was graphed as percentage of the pre-studyCD8+ T cell count in FIGS. 8A and 8B. In cynomolgus monkeys dosed withthe CD20/h2B4 bispecific antibody, Ki67+ T cells increased and peakedbetween day 3 and day 7 post dose, indicating T cell activation.However, in cynomolgus monkeys dosed with NNC/h2B4, there was noincrease in Ki67+ T cells.

Example 12: Flow Cytometry of Human Anti-CD3 Bispecific Antibodies on BCells

This example demonstrates the effect of anti-CD3 arm affinity on B celldepletion.

Cynomolgus monkey studies were conducted and efficacy was determined bymeasuring T cells in peripheral blood by flow cytometry as described inExample 10. Bispecific antibodies were made with an anti-CD20 arm pairedwith 4 anti-CD3 antibody arms with different affinities. Following asingle, i.v. dose at 0.2 ug/kg, efficacy was determined by measuring Bcells in peripheral blood by flow cytometry.

In FIGS. 9A-9D, the resulting B cell count was graphed as a percentageof the pre-study B cell count. Efficacy of B cell depletion correlatesto anti-CD3 arm affinity.

Example 13: In Vitro Study of the Bispecific Antibody on T-Cell MediatedKilling of BCMA Positive Cells

This example illustrates the in vitro cytotoxicity of the Anti-BCMA/CD3hIgG2ΔA Bispecific in BCMA Positive Cells.

Human anti-BCMA (P5A2, A02_Rd4_0.6 nM_C01, A02_Rd4_6 nM_C16, P5C1,C01_Rd4_6 nM_C12, COMBO_Rd4_0.6 nM_C22, Combo_Rd4_0.6 nM_C29, L3PY/H3TAQand A02_Rd4_6 nM_C01) and human anti-CD3 (H2B4) antibodies wereexpressed as human IgG2dA engineered with EEEE for bispecific exchangeas described in Example 8.

CD3+ T cells from PBMC were negatively selected using Pan T CellIsolation kit, human (Miltenyi, San Diego Calif.). Target expressing(KMS12PE, L363 and Molp8) cells and CD3+ T-cells were seeded on clearU-bottom plates at 20000 and 100000 cells/well respectively. Cells weretreated with 10-fold serially diluted bispecific antibody intriplicates. Cell death was determined by CytoTox 96® Non-RadioactiveCytotoxicity Assay (Promega, Madison Wis.) 20 hours after treatment.Cell cytotoxicity was determined as percentage of untreated effectorplus target control wells. EC50 was calculated by Prism software. Table10 shows that all human anti-BCMA_H2B4 bispecific antibodies exert cellkilling activity in BCMA expressing cells.

TABLE 10 KMS12PE KMS12BM MOLP8 Anti-BCMA bispecific (BCMA+++) (BCMA+)(MOLP8+) P5A2 0.371 1.509 5.231 A02_Rd4_0.6nM_C01 0.073 0.078 0.550A02_Rd4_6nM_C16 0.186 0.052 0.315 P5C1 0.581 4.117 N/A C01_Rd4_6nM_C120.189 0.415 0.850 COMBO_Rd4_0.6nM_C22 0.115 0.049 0.065Combo_Rd4_0.6nM_C29 0.175 0.049 0.201 L3PY/H3TAQ 0.070 0.055 0.337A02_Rd4_6nM_C01 UND 0.057 0.060 UND is undetermined; N/A is EC50 couldnot be determined.

Example 14: In Vitro Characterization of the Mouse Hybridoma ClonedAnti-CD3 Antibody

This example illustrates the in vitro T cell activation/proliferation ofthe anti-CD3 cloned from mouse hybridoma in human/cynomolgus PBMC cellsfor antibody screening.

Human anti-CD3 antibodies were cloned from immunized mouse, expressed asmouse IgG1, and purified by Protein A affinity beads. Human/Cynomolgusperipheral blood mononuclear cells (hu/cyPBMC) were prepared by Ficoll(Density: 1.083 g/mL, GE) density gradient centrifugation from bloodfilters obtained from local blood banks. Erythrocytes were removed byincubating in LCK buffer (155 mM NH4Cl, 10 mM KHCO3, 100 mM EDTA; Gibco)for 3 minutes at room temperature. Cells were centrifuged for 5 min at600 g. The supernatant containing the lysed erythrocytes was discarded,and the PBMC were washed twice in 50 ml 1×PBS/1% BSA/1 mM EDTA. Thepelleted cells were adjusted to 10⁷ cells per ml in culture media,X-VIVO-15, serum free media (Lonza), and the PBMC were seeded as 10⁶(100 ul) per-well to round bottom 96 well tissue culture plates.Selected Abs are 10× serial diluted from 1000 ng to 1 ng per mL formixing with human PBMC and 5× serial diluted from 5000 ng to 200 ng permL for mixing with cynomolgus PBMC. For analysis of PBMC T cellproliferation by ³H-thymidine incorporation, 2 day cultures wereperformed in triplicate. During the final 16 h of culture³H-thymidine(0.5 mCi/well) was added, and incorporation was measured. Cells areharvested and lysed, DNA is captured onto glass-fiber filter.Radioactivity (cpm) as measure for proliferation by counting on ascintillation beta-counter.

FIGS. 10A and 10B show that the selected anti-CD3 1A4, 1C10, 2B4, and7A3 antibodies had Thymidine incorporation reading on human andcynomolgus PBMC (peripheral blood mononuclear cells). Table 11 showstheir KDs by Biacore measurement. In vitro characterization showsanti-CD3 1C10 and 2B4 antibodies are similar to the positive controlSP34 anti-CD3 antibody (BD Biosciences)

TABLE 11 anti-xCD3e ab/bsc_hCD3ed kinetic results for data fitted from80 nM-0.64 nM Ligand ka kd t_(1/2) (min) KD (nM) UCHT1(+) 1.80E+05<8.55E−04 >13.5 <4.74 2B4 3.74E+05 2.74E−03 4.21 7.33 1C10 2.96E+052.37E−03 4.88 8.00 SP34(+) 2.84E+05 3.04E−03 3.80 10.73 7A3* 82.70 1A4*99.97 Note: Data is only reported for satisfactory kinetic fits. (+) =positive controls *Kinetic determinations are rough estimates becauseantibody is heterogeneous. Only steady state affinity is measured

Example 15: In Vitro Study of the Bispecific Antibody on T Cell MediatedKilling

This example illustrates the in vitro cytotoxicity of the anti-EpCam/CD3Bispecific in SW480 mixed with healthy donor isolated Pan T cells.

A: Anti-CD Antibodies h2B4-1d, TK, hnpsTK, and yaesTK

Human anti-CD3 (h2B4-1d (or h2B4), h2B4-TK (or h2B4-VH-wt VL_TK),h2B4-hnpsTK (or h2B4-VH-hnps VL_TK), and h2B4-yaesTK (or h2B4-VH-yaesVL_TK)) antibodies and human anti-EpCam antibodies were expressed ashuman IgG2dA engineered with RRRR or EEEE for bispecific exchange asdescribe in Example 8.

The SW480 was selected as target cell line for cell killing assay andthe effector cells and human T cells were purified from human peripheralblood mononuclear cells (huPBMC). Target and effector cells were seededin 96-well, round bottom plates in cell culture medium containing 5%fetal bovine serum (FBS). The number of target cells was kept constantat 2×10⁴ cells/well. A 10-fold serial dilution of bispecific antibody,from 3 ug to 3 pg per mL, was added in triplicate to the cells. Totalreaction volume was 200 uL. The reactions were incubated for 48 and 72hours. For the analysis of cytotoxicity, the lactate dehydrogenase(LDH), a stable cytosolic enzyme that was released upon cell lysis, wasquantitatively measured by CytoTox 96® Non-Radioactive CytotoxicityAssay kit (Promega, G1780). The plate was read on a Vmax kineticmicroplate reader (Molecular Devices) at 490 nM. Optical density valueswere corrected for media background and spontaneous lysis of target andeffector cells. Specific cytotoxicity was calculated according to thefollowing formula:[% spec.lysis=490 nM readout of sample−of E+T mixed control)/(490 nMreadout of total T lysis−of media control)×100%]FIGS. 11A and 11B show that all human anti-EpCam_h2B4 bispecificantibodies had cell killing activity on in vitro setting, and antibodymediated T cell activation was monitoring by T cell activation marker.Table 12A shows their EC50. Table 12B shows Biacore KD on the bispecificantibody format.

TABLE 12A Day2: EC50 (nM) h2B4-1d_Ep 537.1 h2B4-TK_Ep 405.3h2B4-hnpsTK_Ep 424.7 h2B4-yaesTK_Ep 1126

TABLE 12B Summary Table for anti-CD3 hIgG2dA bispecifics kinetics at 37°C. Sample ID - bschIgG2dA ka (1/Ms) kd (1/s) t½ (min) KD (nM) h2B4-1d_Ep5.86E+05 2.37E−02 0.49 40.4 h2B4-TK_Ep 6.87E+05 2.13E−02 0.54 31.0h2B4-hnpsTK_Ep 7.54E+05 2.35E−02 0.49 31.2 h2B4-yaesTK_Ep 4.74E+052.58E−02 0.45 54.4B: Anti-CD antibodies m25A8, h25A8-B12, and h25A8-B13

Human anti-CD3 h2B4(h2B4_1 d) and h25A8 (m25A8, h25A8-B12, andh25A8-B13) and human anti-EpCam antibodies were expressed as humanIgG2dA engineered with RRRR or EEEE for bispecific exchange as describein Example 8.

FIGS. 11C and 11D show that all human anti-EpCam_anti-CD3 bispecificantibodies had cell killing activity on in vitro setting, and antibodymediated T cell activation was monitoring by T cell activation marker.Table 12C shows the EC50 of in vitro cell killing. Table 12D shows theBiacore kinetics on the bispecific antibody format at 37° C. Table 12Eshows in vitro characterization using SEC-MALS (Size ExclusionChromatography with Multi-Angle Light Scattering) and DSC (DifferentialScanning Calorimety).

TABLE 12C EC50 of in vitro cell killing Day 1: EC50 (nM) h2B4-1d_Ep 52.9m25A8_Ep 127.6 h25A8-B12_Ep 99.36 h25A8-B13_Ep 57.11

TABLE 12D Summary Table for anti-CD3 hIgG2dA bispecifics kinetics at 37°C. SampleID - bschIgG2dA ka (1/Ms) kd (1/s) t1/2 (min) KD (nM)h25A8-B5_Ep 1.29E+06 5.81E−02 0.20 45.0 h25A8-B8_Ep 1.19E+06 2.22E−020.52 18.7 h25A8-B12_Ep 1.15E+06 2.41E−02 0.48 21.0 h25A8-B13_Ep 1.20E+062.19E−02 0.53 18.3 h25A8-C8_Ep 1.20E+06 3.01E−02 0.38 25.1 h2B4-1d_Ep5.86E+05 2.37E−02 0.49 40.4

TABLE 12E in vitro characterization SEC-MALS DSC % % Tm1 C Tm2 C Tm3 CMonomer Aggregate (CH2) (Fab) (CH3) m28A8 99.9 0.1 66.3 67.8 76.1hIgG2dA_h25A8- 99.2 0.8 70.91 73.06 78.31 B12 hIgG2dA_h25A8- 99.5 0.570.88 72.86 78.2 B13 hIgG2dA-h2B4TK 98.8 1.4 70.2 74.5 79.4

Example 16: In Vitro Study of the Bispecific Antibody on T Cell MediatedKilling of Primary Myeloma Patient Samples

This example illustrates the in vitro cytotoxicity of the anti-BCMA/CD3Bispecific in primary myeloma cells.

Human anti-BCMA (P5A2, A02_Rd4_0.6 nM_C01, A02_Rd4_6 nM_C16,Combo_Rd4_0.6 nM_C29, and P6E01 L3PY/H3TAQ) and human anti-CD3 (h2B4)antibodies were expressed as human IgG2dA engineered with EEEE or RRRRfor bispecific exchange as describe in Example 8.

Total bone marrow mononuclear cells from myeloma patients were seeded inclear U-bottom plates at total bone marrow mononuclear cell numbers thatresulted in 3000-5000 myeloma cells/well. Cells were treated with10-fold serially diluted bispecific antibody. Five days after treatment,total viable cells were determined by flow cytometry using antibodies toCD138 and CD38 (Biolegend, CA). Cells were incubated with antibodies at4° in PBS+0.5% FBS for 30 minutes. Cells were washed and FixableViability Dye eFluor 780 (eBioscience, Inc., CA) in PBS was added to thecells for 30 minutes at 4°. Prior to cell acquisition on a BD flowcytometer, cells were washed and CountBright Absolute Counting Beads(Molecular Probes, OR) were added. Percent live cells were determined aslive cell count in treated vs untreated wells using counting beads. EC50was calculated by Prism software.

Table 13A shows that all human anti-BCMA_h2B4 bispecific antibodies havecell killing activity on myeloma patient samples and patient T cells arefunctional effector cells. Table 13B shows killing of one anti-BCMAbispecific on multiple myeloma patient samples with different effectorto target (E:T) ratio.

TABLE 13A Anti-BCMA bispecific EC50 (Patient MM00146) (nM) P6E01L3PY/H3TAQ 0.015 P5A2 0.946 A02_Rd4_0.6nM_C01 0.064 A02_Rd4_6nM_C160.029 NNC_2b41d hIgG2dA 3.302

TABLE 13B Combo_Rd4_0.6nM_C29 Patient EC50 (nM) E:T MM00146 0.035 1:1MM00147 0.031 2:1 MM00151 0.02 3:1 MM00152 0.289 1:2

Example 17: ELISPOT of Antibody Secreting Cells from Cynomolgus MonkeysAdministered with Anti-BCMA/CD3 Bispecific Antibodies

This example illustrates the depletion of IgG secreting cells incynomolgus monkey with anti-BCMA/CD3 bispecific antibodies.

Cynomolgus monkeys (n=2) were dosed via intravenous bolus injection withbispecific anti-BCMA_CD3 antibodies (h2B4-VH-hnps VL_TK), A02_Rd4_0.6nM_C01/H2B4 and Combo_Rd4_0.6 nM_C29/H2B at two doses, day 1 and day 8,of 100 ug/kg and 300 ug/kg. Animals were observed twice daily and ateach blood collection time point. Peripheral blood mononuclear cells(PBMC) were sampled on days −6, 4 and 10. Bone marrow samples were takenon day 10 when animals were necropsied.

Blood was collected into Becton Dickinson® CPT™ Cell Preparation Tubescontaining sodium heparin and a density gradient, and then PBMCs werecollected at the gradient interface following centrifugation.

A sample of bone marrow (from femur) was collected by flushing withapproximately 5 mL of 100% fetal bovine serum (FBS) and then single cellsuspensions were prepared by suspending flushed marrow in 50 ml ofbuffer.

Cells were counted using the Cellometer Vision and adjusted withcomplete RPMI 1640 culture medium to a concentration of 5×10⁶ cells permL for PBMC and 2×10⁶ cells per mL for bone marrow cells. TotalIgG-secreting cells were enumerated using the ELISpot^(BASIC) kit fromMabtech (#3850-2HW-Plus). Briefly, PBMC or bone marrow cells were addedto triplicate wells at specified concentrations (PBMCs at 5×10⁵/well,and bone marrow cells at 2×10⁵/well), and then cells were seriallydiluted in the plate. After an overnight incubation, plates were washedand a biotinylated detection antibody was added. Plates were incubatedfor 2 hours and streptavidin-HRP was added for 1 hour. IgG spots werevisualized using TMB substrate solution and counted using the ImmunoSpotImaging Analyzer system (CTL) and ImmunoSpot 5.1 software. Data wereexpressed as the mean (+/−SD) number of IgG-secreting cells fromtriplicate samples.

The resulting IgG-secreting cell count in PBMC and in bone marrow arelisted in Tables 14A and 14B, respectively. Depletion of IgG-secretingcells was seen for both anti-BCMA/CD3 bispecific antibodies in PBMC ascompared to pre-dose and bone marrow as compared to vehicle and negativecontrol. A dose dependent effect was seen in the bone marrow.

TABLE 14A Day PBMC −6 4 10 Vehicle 204, 320 323, 538 127, 137 0.1 mg/kg107, 128 5, 0 0, 0 Combo_Rd4_0.6nM_C29 0.3 mg/kg 304, 362 0, 5 0, 0Combo_Rd4_0.6nM_C29 0.1 mg/kg 447, 672 16, 27 21, 10 A02_Rd4_0.6nM_C010.3 mg/kg 512, 224 25, 8 0, 0 A02_Rd4_0.6nM_C01 0.3 mg/kg NNC_2b41d 139,361 1345, 1154 992, 928

TABLE 14B Day Bone Marrow 10 Vehicle 2614, 8093 0.1 mg/kgCombo_Rd4_0.6nM_C29 35, 18 0.3 mg/kg Combo_Rd4_0.6nM_C29 35, 22 0.1mg/kg A02_Rd4_0.6nM_C01 996, 960 0.3 mg/kg A02_Rd4_0.6nM_C01 2170, 930.3 mg/kg NNC_2b41d 5980, 2893

Example 18: Anti-BCMA/CD3 Bispecific Induce Tumor Regression andInhibition in MM1.S Tumor Model

This example illustrates tumor regression and inhibition in anorthotopic MM1.S myeloma model.

In vivo efficacy study of BCMA bispecifics was performed with MM1.S,expressing luciferase and GFP, orthotopic model. One day prior to tumorcell injection, mice were irradiated with 100 cGy using RS 2000Biological Research Irradiator (RAD Source Technolgies, GA). Fivemillion MM1.S LucGFP cells were injected intravenously through the tailvein into 6-8 weeks old female Nod/Scid/IL2Rg^(−/−) (NSG) animals.Intraperitoneal injection of D-luciferin (Regis Technologies, MortonGrove, Ill.) (200 uL per animal at 15 mg/mL), followed by anesthesiawith isofluorane and subsequent whole body bioluminescence imaging (BLI)enabled monitoring of tumor burden. Bioluminescent signals emitted bythe interaction between luciferase expressed by the tumor cells andluciferin were captured by imaging using an IVIS Spectrum CT (PerkinElmer, MA) and quantified as total flux (photons/sec) using Living Image4.4 (Caliper Life Sciences, Alameda, Calif.). When the total fluxreached an average of 15E6 for all animals, the animals were injectedthrough bolus tail vein with 20 million expanded T cells from PBMC.Briefly, pan-T cells purchased from AlICells (Alameda, Calif.) wereactivated with human T Cell Activation/Expansion Kit (Miltenyi, SanDiego, Calif.). After three days, 15 U/mL of IL2 (ebioscience, SanDiego, Calif.) was added every two days until day 11. Cells wereharvested, activation/expansion beads were magnetically removed, andcells were washed and resuspended in PBS. One day post T cell injection,mice were imaged as described above and animals were randomized intogroups of seven mice; A02_Rd4_0.6 nM_C01 at 0.03 mg/kg and 0.3 mg/kg andCombo_Rd4_0.6 nM_C29 at 0.03 mg/kg, 0.1 mg/kg and 0.3 mg/kg. A singledose of human anti-BCMA/CD3 (h2B4-VH-wt VL_TK) bispecific and negative(NNC) control bispecific antibody was administered through bolus tailvein injection. Animals were sacrificed when they exhibited hindlimbparalysis, an endpoint for MM1.S orthotopic model. FIG. 12 shows that asingle dose of human anti-BCMA/CD3 bispecific antibody resulted in tumorregression in a dose-dependent manner.

Example 19: Two Doses of Anti-BCMA/CD3 Bispecific Induce TumorRegression in Aggressive Molp8 Tumor Model

This example illustrates tumor regression with two doses ofanti-BCMA/CD3 bispecific antibodies in an orthotopic Molp8 myelomamodel.

In vivo efficacy study of BCMA bispecifics was performed with Molp8,expressing luciferase and GFP, orthotopic model. Two million Molp8LucGFP cells were injected intravenously through the tail vein into 6-8weeks old female NSG animals. Intraperitoneal injection of D-luciferin(Regis Technologies, Morton Grove, Ill.) (200 uL per animal at 15mg/mL), followed by anesthesia with isofluorane and subsequent wholebody bioluminescence imaging (BLI) enabled monitoring of tumor burden.Bioluminescent signals emitted by the interaction between luciferaseexpressed by the tumor cells and luciferin were captured by imagingusing an IVIS Spectrum CT (Perkin Elmer, MA) and quantified as totalflux (photons/sec) using Living Image 4.4 (Caliper Life Sciences,Alameda, Calif.). When the total flux reached an average of 25E6 for allanimals were randomized into three groups of seven mice; 1)Combo_Rd4_0.6 nM_C29, 0.3 mg/kg, 2) Combo_Rd4_0.6 nM_C29, 0.3 mg/kg, twodoses and 3) NNC_2 B4, 0.3 mg/kg, two doses. The animals were injectedthrough bolus tail vein with 20 million expanded T cells as described inExample 18. Two days post T cell injection, mice were dosed withbispecific antibodies. Animals were sacrificed when they exhibitedweight loss of more than 15%, an endpoint for Molp8 orthotopic model.FIG. 13 shows that two doses of human anti-BCMA/CD3 (h2B4-VH-wt VL_TK)bispecific antibody resulted in increased tumor regression.

Example 20: Anti-BCMA/CD3 Bispecific in Combination with Standard ofCare for Multiple Myeloma in Orthotopic Molp8 Tumor Model

This example demonstrates no opposing effects on anti-BCMA/CD3bispecific antibodies when combined with bortezomib or lenalidomide andbetter potency with anti-BCMA/CD3 bispecific antibodies as compared tobortezomib and lenalidomie combined.

In vivo efficacy study of BCMA bispecifics was performed with Molp8,expressing luciferase and GFP, orthotopic model. Two million Molp8LucGFP cells were injected intravenously through the tail vein into 6-8weeks old female NSG animals. Intraperitoneal injection of D-luciferin(Regis Technologies, Morton Grove, Ill.) (200 uL per animal at 15mg/mL), followed by anesthesia with isofluorane and subsequent wholebody bioluminescence imaging (BLI) enabled monitoring of tumor burden.Bioluminescent signals emitted by the interaction between luciferaseexpressed by the tumor cells and luciferin were captured by imagingusing an IVIS Spectrum CT (Perkin Elmer, MA) and quantified as totalflux (photons/sec) using Living Image 4.4 (Caliper Life Sciences,Alameda, Calif.). The animals were injected through bolus tail vein onday 7 with 20 million expanded T cells as described in Example 18. Twodays post T cell injection, mice were imaged and randomized into fivegroups of seven mice with an average of 17E6 total flux/group: 1)Combo_Rd4_0.6 nM_C29, 0.3 mg/kg, 2) Combo_Rd4_0.6 nM_C29, 0.3 mg/kg and1 mg/kg bortezomib, 3) Combo_Rd4_0.6 nM_C29, 0.3 mg/kg and 50 mg/kglenalidomide, 4) 1 mg/kg bortezomib and 50 mg/kg lenalidomide and 5)vehicle. Anti-BCMA/CD3 (in PBS) was injected through bolus tail veininjection, bortezomib (in PBS) was administrated via intraperitonealinjection and lenalidomide (30% PEG400/5% propylene glycol/0.5% Tween80)via oral gavage. Vehicle consisted of 30% PEG400/5% propyleneglycol/0.5% Tween80, which was administrated via oral gavage. Animalswere sacrificed when they exhibit weight loss of more than 15%, anendpoint for Molp8 orthotopic model. FIG. 14 shows that combining ananti-BCMA/CD3 (h2B4-VH-hnps VL-TK) bispecific antibody with bortezomibor lenalidomide did not have a negative effect on the efficacy of theanti-BCMA/CD3 bispecific antibody. In this model, anti-BCMA/CD3bispecific antibody alone or in combination with lenalidomide orbortezomib is more efficacious than lenalidomide and bortezomibcombined.

Example 21: In Vitro Study of Anti-BCMA/CD3 Bispecific in Combinationwith Lenalidomide, Carfilzomib or Doxorubicin on OPM2 Cell Line

This example illustrates no adverse effects on T cell function whencombined with carfilzomib, doxorubicin, and lenalidomide foranti-BCMA/CD3 bispecific antibody activity as compared to the bispecificantibody alone.

CD3+ T cells from PBMC were negatively selected using Pan T CellIsolation kit, human (Miltenyi, San Diego Calif.). OPM2 cells and CD3+T-cells were seeded in clear U-bottom plates 20000 and 100000cells/well, respectively. OPM2 and CD3+ T cells were first incubatedwith the standard of care for two hours at 37°. 1.56 nM carfilzomib and6.25 nM doxorubicin were diluted in PBS containing 0.02% DMSO.Lenalidomide was diluted in PBS containing 0.1% DMSO at 195 nM. Cellswere treated with 10-fold serially diluted bispecific antibody. Threedays after treatment, total viable cells were determined by flowcytometry using antibodies to CD138, CD4, and CD8 (Biolegend). Cellswere incubated with antibodies at 4° in PBS+0.5% FBS for 30 minutes.Cells were washed and Fixable Viability Dye eFluor 780 (eBioscience,Inc., CA) in PBS was added to the cells for 30 minutes at 4°. Prior tocell acquisition on a BD flow cytometer, cells were washed andCountBright Absolute Counting Beads (Molecular Probes, OR) were added.Percent live cells were determined as live cell count in treated vsuntreated wells using counting beads. FIGS. 15A, 15B, and 15C,respectively, show that carfilzomib, lenalidomide, and doxorubicin donot have a negative effect on the function of Combo_Rd4_0.6 nM_C29-CD3(h2B4-VH-hnps VL-TK) bispecific antibody on OPM2 cells.

Example 22: In Vitro Study of Anti-BCMA/CD3 Bispecific in Combinationwith Lenalidomide and Carfilzomib on KMS12BM Cell Line

This example illustrates synergistic effects on anti-BCMA/CD3 bispecificfunction when combined with carfilzomib and lenalidomide as compared toeach molecule alone.

CD3+ T cells from PBMC were negatively selected using Pan T CellIsolation kit, human (Miltenyi, San Diego Calif.). KMS12BM cells andCD3+ T-cells were seeded in clear U-bottom plates 20000 and 100000cells/well, respectively. Cells were treated with 0.017 nM anti-BCMA/CD3bispecific in combination with carfilzomib and a range of concentrationfor lenalidomide. 1.25 nM carfilzomib was diluted in PBS containing0.02% DMSO. Lenalidomide was diluted in PBS containing 0.1% DMSOstarting at 4 uM diluted 4-fold. Three days after treatment, totalviable cells were determined by flow cytometry using antibodies toCD138, CD4, and CD8 (Biolegend, CA). Cells were incubated withantibodies at 4° in PBS+0.5% FBS for 30 minutes. Cells were washed andFixable Viability Dye eFluor 780 (eBioscience, Inc., CA) in PBS wasadded to the cells for 30 minutes at 4°. Prior to cell acquisition on aBD flow cytometer, cells were washed and CountBright Absolute CountingBeads (Molecular Probes, OR) were added. Percent live cells weredetermined as live cell count in treated versus untreated wells usingcounting beads. FIG. 16 shows that at the concentrations tested,carfilzomib, lenalidomide, and anti-BCMA/CD3 (h2B4-VH-hnps VL-TK)bispecific antibody had very little single agent cytotoxic function.When all three agents were combined, a synergistic effect was observedat a dose-dependent lenalidomide concentration in KMS12BM cells.

Although the disclosed teachings have been described with reference tovarious applications, methods, kits, and compositions, it will beappreciated that various changes and modifications can be made withoutdeparting from the teachings herein and the claimed invention below. Theforegoing examples are provided to better illustrate the disclosedteachings and are not intended to limit the scope of the teachingspresented herein. While the present teachings have been described interms of these exemplary embodiments, the skilled artisan will readilyunderstand that numerous variations and modifications of these exemplaryembodiments are possible without undue experimentation. All suchvariations and modifications are within the scope of the currentteachings.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The foregoing description and Examples detail certain specificembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

It is claimed:
 1. A method of treating a condition associated withmalignant cells expressing BCMA (B-Cell Maturation Antigen) in a subjectcomprising administering to the subject in need thereof an effectiveamount of a pharmaceutical composition comprising a bispecific antibodycomprising a first antibody variable domain capable of specificallybinding to CD3, and a second antibody variable domain capable ofspecifically binding to BCMA, wherein the first antibody variable domaincomprises a heavy chain variable (VH) region comprising a VH CDR1, VHCDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 320, 322, 324,326, 328, 330, 345, 347, 349, 351, 444, 354, 356, 378, 442, 380, 382,384 386, 388, 390, 392, 394, 396, 398, or 400; and a light chainvariable (VL) region comprising a VL CDR1, VL CDR2, and VL CDR3 of theVL sequence shown in SEQ ID NO: 319, 321, 323, 325, 327, 329, 344, 346,348, 350, 352, 355, 377, 443, 445, 379, 381, 383, 385, 387, 389, 391,393, 395, 397, or
 399. 2. The method of claim 1, wherein the conditionis a cancer.
 3. The method of claim 2, wherein the cancer is a B-cellrelated cancer selecting from the group consisting of multiple myeloma,malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocytepredominant Hodgkin's lymphoma, Kahler's disease and Myelomatosis,plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia,hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloidleukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocyticleukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma,Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, largecell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia,Waldenstrom's macroglobulienemia, diffuse large B cell lymphoma,mucosa-associated lymphatic tissue lymphoma, small cell lymphocyticlymphoma, primary mediastinal (thymic) large B-cell lymphoma,lymphoplasmactyic lymphoma, nodal marginal zone B cell lymphoma, splenicmarginal zone lymphoma, intravascular large B-cell lymphoma, primaryeffusion lymphoma, lymphomatoid granulomatosis, T cell/histiocyte-richlarge B-cell lymphoma, primary central nervous system lymphoma, primarycutaneous diffuse large B-cell lymphoma (leg type), EBV positive diffuselarge B-cell lymphoma of the elderly, diffuse large B-cell lymphomaassociated with inflammation, ALK-positive large B-cell lymphoma,plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associatedmulticentric Castleman disease, B-cell lymphoma unclassified withfeatures intermediate between diffuse large B-cell lymphoma and Burkittlymphoma, B-cell lymphoma unclassified with features intermediatebetween diffuse large B-cell lymphoma and classical Hodgkin lymphoma,and other B-cell related lymphoma.
 4. A method of inhibiting tumorgrowth or progression in a subject who has malignant cells expressingBCMA (B-Cell Maturation Antigen), comprising administering to thesubject in need thereof an effective amount of a pharmaceuticalcomposition comprising a bispecific antibody, wherein the bispecificantibody comprises a first antibody variable domain capable ofspecifically binding to CD3, and a second antibody variable domaincapable of specifically binding to BCMA comprising a heavy chainvariable (VH) region comprising (i) a VH CDR1 comprising the sequenceshown in SEQ ID NO:150, 151, 152, 156, or 157; (ii) a VH CDR2 comprisingthe sequence shown in SEQ ID NO: 169, 154, 194, 159, 195, 196, 162, 158,198, 177, 178, 199, 200, 201, 202, 203, 204, 206, 207, 208, 172, 359, or360; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO:155, 161, 197, 205, 164, or 361; and wherein the light chain variable(VL) region comprises (i) a VL CDR1 comprising the sequence shown in SEQID NO: 209, 271, 273, 275, 251, 277, 260, 279, 245, 283, 285, 287, 290,292, 235, 297, or 299; (ii) a VL CDR2 comprising the sequence shown inSEQ ID NO: 221 or 362; and (iii) a VL CDR3 comprising the sequence shownin SEQ ID NO: 211, 225, 272, 274, 276, 278, 280, 281, 282, 284, 286,288, 289, 291, 293, 294, 229, 296, 298, or
 300. 5. A method ofinhibiting metastasis of malignant cells expressing BCMA in a subject,comprising administering to the subject in need thereof an effectiveamount of a pharmaceutical composition comprising a bispecific antibody,wherein the bispecific antibody comprises a first antibody variabledomain capable of specifically binding to CD3, a second antibodyvariable domain capable of specifically binding to BCMA comprising aheavy chain variable (VH) region comprising (i) a VH CDR1 comprising thesequence shown in SEQ ID NO:150, 151, 152, 156, or 157; (ii) a VH CDR2comprising the sequence shown in SEQ ID NO: 169, 154, 194, 159, 195,196, 162, 158, 198, 177, 178, 199, 200, 201, 202, 203, 204, 206, 207,208, 172, 359, or 360; and (iii) a VH CDR3 comprising the sequence shownin SEQ ID NO: 155, 161, 197, 205, 164, or 361; and wherein the lightchain variable (VL) region comprises (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 209, 271, 273, 275, 251, 277, 260, 279,245, 283, 285, 287, 290, 292, 235, 297, or 299; (ii) a VL CDR2comprising the sequence shown in SEQ ID NO: 221 or 362; and (iii) a VLCDR3 comprising the sequence shown in SEQ ID NO: 211, 225, 272, 274,276, 278, 280, 281, 282, 284, 286, 288, 289, 291, 293, 294, 229, 296,298, or
 300. 6. A method of inducing tumor regression in a subject whohas malignant cells expressing BCMA, comprising administering to thesubject in need thereof an effective amount of a pharmaceuticalcomposition comprising a bispecific antibody, wherein the bispecificantibody comprises a first antibody variable domain capable ofspecifically binding to CD3, and a second antibody variable domaincapable of specifically binding to BCMA comprising a heavy chainvariable (VH) region comprising (i) a VH CDR1 comprising the sequenceshown in SEQ ID NO:150, 151, 152, 156, or 157; (ii) a VH CDR2 comprisingthe sequence shown in SEQ ID NO: 169, 154, 194, 159, 195, 196, 162, 158,198, 177, 178, 199, 200, 201, 202, 203, 204, 206, 207, 208, 172, 359, or360; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO:155, 161, 197, 205, 164, or 361; and wherein the light chain variable(VL) region comprises (i) a VL CDR1 comprising the sequence shown in SEQID NO: 209, 271, 273, 275, 251, 277, 260, 279, 245, 283, 285, 287, 290,292, 235, 297, or 299; (ii) a VL CDR2 comprising the sequence shown inSEQ ID NO: 221 or 362; and (iii) a VL CDR3 comprising the sequence shownin SEQ ID NO: 211, 225, 272, 274, 276, 278, 280, 281, 282, 284, 286,288, 289, 291, 293, 294, 229, 296, 298, or
 300. 7. A method of treatingmultiple myeloma in a subject in need thereof, comprising administeringto the subject an effective amount of a pharmaceutical compositioncomprising a bispecific antibody and one or more other therapeuticagents selected from the group consisting of bortezomib, lenalidomide,carfilzomib, and doxorubicin, wherein the bispecific antibody comprisesa first antibody variable domain capable of specifically binding to CD3,and a second antibody variable domain capable of specifically binding toBCMA comprising a heavy chain variable (VH) region comprising (i) a VHCDR1 comprising the sequence shown in SEQ ID NO:150, 151, 152, 156, or157; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 169,154, 194, 159, 195, 196, 162, 158, 198, 177, 178, 199, 200, 201, 202,203, 204, 206, 207, 208, 172, 359, or 360; and (iii) a VH CDR3comprising the sequence shown in SEQ ID NO: 155, 161, 197, 205, 164, or361; and wherein the light chain variable (VL) region comprises (i) a VLCDR1 comprising the sequence shown in SEQ ID NO: 209, 271, 273, 275,251, 277, 260, 279, 245, 283, 285, 287, 290, 292, 235, 297, or 299; (ii)a VL CDR2 comprising the sequence shown in SEQ ID NO: 221 or 362; and(iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 211, 225,272, 274, 276, 278, 280, 281, 282, 284, 286, 288, 289, 291, 293, 294,229, 296, 298, or
 300. 8. The method of claim 1, wherein the secondantibody variable domain comprises a heavy chain variable (VH) regioncomprises (i) a VH CDR1 comprising the sequence shown in SEQ ID NO:150,151, 152, 156, or 157; (ii) a VH CDR2 comprising the sequence shown inSEQ ID NO: 169, 154, 194, 159, 195, 196, 162, 158, 198, 177, 178, 199,200, 201, 202, 203, 204, 206, 207, 208, 172, 359, or 360; and (iii) a VHCDR3 comprising the sequence shown in SEQ ID NO: 155, 161, 197, 205,164, or 361; and wherein the light chain variable (VL) region comprises(i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 209, 271, 273,275, 251, 277, 260, 279, 245, 283, 285, 287, 290, 292, 235, 297, or 299;(ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 221 or 362;and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 211,225, 272, 274, 276, 278, 280, 281, 282, 284, 286, 288, 289, 291, 293,294, 229, 296, 298, or
 300. 9. The method of claim 8, wherein the (a)first antibody variable domain comprises a heavy chain variable (VH)region comprising (i) a VH complementarity determining region one (CDR1)comprising the sequence shown in SEQ ID NO: 331, 332, 333, 401, 407, or408; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 336,417, 404, or 405; and iii) a VH CDR3 comprising the sequence shown inSEQ ID NO: 335 or 406; and a light chain variable (VL) region comprising(i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 343 or 441;(ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 341 or 436;and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 342 or439; and the (b) the second antibody variable domain comprises a heavychain VH region comprising a heavy chain variable (VH) region comprising(i) a VH CDR1 comprising the sequence shown in SEQ ID NO: 151, 156, or157; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 158 or159; and (iii) a VH CDR3 comprising SEQ ID NO: 155; and wherein thelight chain variable (VL) region comprises (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 209; (ii) a VL CDR2 comprising the sequenceshown in SEQ ID NO: 221; and (iii) a VL CDR3 comprising the sequenceshown in SEQ ID NO:
 225. 10. The method of claim 4, wherein the (a)first antibody variable domain comprises a heavy chain variable (VH)region comprising (i) a VH complementarity determining region one (CDR1)comprising the sequence shown in SEQ ID NO: 331, 332, 333, 401, 407, or408; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 336,417, 404, or 405; and iii) a VH CDR3 comprising the sequence shown inSEQ ID NO: 335 or 406; and a light chain variable (VL) region comprising(i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 343 or 441;(ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 341 or 436;and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 342 or439; and the (b) the second antibody variable domain comprises a heavychain VH region comprising a heavy chain variable (VH) region comprising(i) a VH CDR1 comprising the sequence shown in SEQ ID NO: 151, 156, or157; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 158 or159; and (iii) a VH CDR3 comprising SEQ ID NO: 155; and wherein thelight chain variable (VL) region comprises (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 209; (ii) a VL CDR2 comprising the sequenceshown in SEQ ID NO: 221; and (iii) a VL CDR3 comprising the sequenceshown in SEQ ID NO:
 225. 11. The method of claim 5, wherein the (a)first antibody variable domain comprises a heavy chain variable (VH)region comprising (i) a VH complementarity determining region one (CDR1)comprising the sequence shown in SEQ ID NO: 331, 332, 333, 401, 407, or408; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 336,417, 404, or 405; and iii) a VH CDR3 comprising the sequence shown inSEQ ID NO: 335 or 406; and a light chain variable (VL) region comprising(i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 343 or 441;(ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 341 or 436;and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 342 or439; and the (b) the second antibody variable domain comprises a heavychain VH region comprising a heavy chain variable (VH) region comprising(i) a VH CDR1 comprising the sequence shown in SEQ ID NO: 151, 156, or157; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 158 or159; and (iii) a VH CDR3 comprising SEQ ID NO: 155; and wherein thelight chain variable (VL) region comprises (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 209; (ii) a VL CDR2 comprising the sequenceshown in SEQ ID NO: 221; and (iii) a VL CDR3 comprising the sequenceshown in SEQ ID NO:
 225. 12. The method of claim 6, wherein the (a)first antibody variable domain comprises a heavy chain variable (VH)region comprising (i) a VH complementarity determining region one (CDR1)comprising the sequence shown in SEQ ID NO: 331, 332, 333, 401, 407, or408; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 336,417, 404, or 405; and iii) a VH CDR3 comprising the sequence shown inSEQ ID NO: 335 or 406; and a light chain variable (VL) region comprising(i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 343 or 441;(ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 341 or 436;and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 342 or439; and the (b) the second antibody variable domain comprises a heavychain VH region comprising a heavy chain variable (VH) region comprising(i) a VH CDR1 comprising the sequence shown in SEQ ID NO: 151, 156, or157; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 158 or159; and (iii) a VH CDR3 comprising SEQ ID NO: 155; and wherein thelight chain variable (VL) region comprises (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 209; (ii) a VL CDR2 comprising the sequenceshown in SEQ ID NO: 221; and (iii) a VL CDR3 comprising the sequenceshown in SEQ ID NO:
 225. 13. The method of claim 7, wherein the (a)first antibody variable domain comprises a heavy chain variable (VH)region comprising (i) a VH complementarity determining region one (CDR1)comprising the sequence shown in SEQ ID NO: 331, 332, 333, 401, 407, or408; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 336,417, 404, or 405; and iii) a VH CDR3 comprising the sequence shown inSEQ ID NO: 335 or 406; and a light chain variable (VL) region comprising(i) a VL CDR1 comprising the sequence shown in SEQ ID NO: 343 or 441;(ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 341 or 436;and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO: 342 or439; and the (b) the second antibody variable domain comprises a heavychain VH region comprising a heavy chain variable (VH) region comprising(i) a VH CDR1 comprising the sequence shown in SEQ ID NO: 151, 156, or157; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 158 or159; and (iii) a VH CDR3 comprising SEQ ID NO: 155; and wherein thelight chain variable (VL) region comprises (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 209; (ii) a VL CDR2 comprising the sequenceshown in SEQ ID NO: 221; and (iii) a VL CDR3 comprising the sequenceshown in SEQ ID NO: 225.